Compounds and methods for modulating pharmacokinetics

ABSTRACT

Methods and compositions are described for management of the pharmacokinetic properties of active agents, e.g., therapeutic moieties, by conjugating, fusing, or non-direct linkage of the active agent to one or more wild-type or modified heparin-binding peptides (HB). Compounds may be administered to tissues including skin. Contemplated uses include treatment of disease, allergen immunotherapy, and immunization. Other aspects relate to compositions, methods and kits comprising heparin-binding peptides (HB) fused or conjugated to the therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 61/948,724, filed Mar. 6, 2014, the contents of whichare incorporated by reference. This application is acontinuation-in-part of U.S. patent application Ser. No. 14/409,270,filed Dec. 18, 2014, which is the national stage entry ofPCT/US2013/047550, filed Jun. 25, 2013, which claims priority to and thebenefit of U.S. Provisional Application No. 61/663,679 filed on Jun. 25,2012, the contents of each of which are incorporated by reference.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 17, 2015, isnamed 043214-073189-CIP_SL.txt and is 65,901 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions tomanage the release profile of active agents, e.g., therapeutic agents,by conjugating, fusing, or non-direct linkage of the active agent to oneor more heparin-binding peptides (HB). Other aspects relate tocompositions, methods and kits comprising heparin-binding peptides (HB)fused or conjugated to the therapeutic agents.

BACKGROUND

Many promising disease therapies are presently hampered by a shorthalf-life in the systemic circulation. In order to be effective, thesetherapies may require frequent injections and high dosages which cancause discomfort, inconvenience, and potentially severe adversereactions. In some cases, these issues may prevent an otherwisebeneficial therapy from being used, and afflicted individuals are leftto suffer.

The inability to effectively manage the pharmacokinetics of therapeuticmolecules causes problems in a number of areas including vaccinations,allergies, and the development of therapeutic proteins and other drugs.

Many vaccines require a multiple doses to develop complete, long-termimmunity. Vaccines may require a series of injections over time. Thiscan prove especially problematic for young children who are commonly therecipients of vaccines. The inconvenience and discomfort associated withmulti-dose vaccines may lead to non-compliance, potentially negating notonly personal immunity, but many of the large scale disease controlbenefits associated with widespread vaccination as well.

The developing field of allergen immunotherapy involves inducing anallergen tolerance in a patient through managed exposure to theallergen. While many people suffering from a variety of potentiallyserious allergies could benefit from this treatment, it requiresmaintaining prolonged exposure to small quantities of allergens which incertain instances can involve weekly or monthly injections for fiveyears or more or sublingual application as often as daily for up to fiveyears.

In the area of protein therapeutics, fast renal clearance and rapiddegradation lead to short half-lives in systemic circulation, limitingthe usefulness of many compounds that may otherwise benefit patients.

SUMMARY

The invention relates to controlled delivery of compounds usingheparin-binding (HB) domains. By linking therapeutic moieties to one ormore HB domains, retention in tissue and, therefore, systemic release ofthe therapeutic moiety may be controlled. The pharmacokinetics of thecompounds may be modified by, for example, adding or subtracting HBdomains or modifying the amino acid sequence of the one or more HBdomains. By controlling the retention of therapeutic moieties in tissue,and therefore the release of those moieties into systemic circulation,compounds of the invention may enable the therapeutic use of otherwisepromising protein drugs that suffer from rapid degradation. Compounds ofthe system may also lead to fewer, less-frequent, and lowerconcentration doses of compounds with applications includingvaccinations/immunizations and allergen immunotherapy.

The present embodiments provide for the controlled release ofrecombinant therapeutic proteins or small molecules to cells or tissuesthat express proteoglycans including, but not limited to cartilage,brain and spinal cord tissue, skin and subcutaneous tissue. Morespecifically, some embodiments herein are directed to novelheparin-binding peptides (HB) fused to a therapeutic protein or aportion thereof, optionally by a linker peptide. Some embodimentsprovide for the conjugation or indirect linkage of small molecules tonovel HB for controlled release. The HB-agent compositions can be usedin tandem for delivery of therapeutic proteins and agents to tissues.

An aspect of the present invention provides for a controlled releasetherapeutic composition comprising, for example,(HB-linker)_(n)-Xm-(linker-HB)o, where HB is a heparin binding protein,X is an active agents such as a therapeutic protein or a portionthereof, or a therapeutic small molecule, and where n, m, and o areintegers, and m is at least one and n+o is at least one. In someembodiments, a HB-X conjugate is HB-Xn, or (HB-linker)n-Xn, and where nis an integer of at least 1. In some embodiments, the composition is arecombinant fusion protein comprising a recombinant HB and thetherapeutic protein (or portion thereof). The components of thecomposition can be placed in order, relative to the N-terminus of the HBportion of the composition: HB-X, X-HB, HB-linker-X, (HB-linker)2-X,X-linker-HB, X-HB-X, HBn-X-HBn, (HB-linker)n—X-(linker-HB)n, HB-X-HB-X,etc. Additionally, the composition can comprise a mixture of HB-Xconstructs, wherein X represents different proteins or small molecules(i.e., a composition comprising HB-X1 and HB-X2, etc.). An examplelinker is a peptide comprising the amino acids GGG. Other linkerscommonly known in the art are encompassed for use in the HB-X conjugate,such as known peptide linkers and chemical linkers.

More specifically, the HB portion of the composition is positivelycharged through many lysine and arginine residues in the HB peptide,which binds to cellular or tissue expressing proteoglycans which arenegatively charged by sulfate groups. In particular embodiments, the HBis mutated by replacing the native cysteine residue with an arginine orlysine residue. This may enhance positive charge but it also appears toprovide a benefit beyond what would be expected solely on the change incharge. The HB domain may be at least 80% similar toKRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1) and is preferably at least 90%similar. Specific examples of a suitable HB domain include thoseselected from the following peptides having the amino acid residuesequences: KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1); KRKKKGKGLGKKRDPRLRKYK(SEQ ID NO: 2) (also referred to as HB C16R); KRKKKGKGLGKKRDPSLRKYK (SEQID NO: 82) (also referred to as HB C16S); or KRKKKGKGLGKKRDPKLRKYK (SEQID NO: 3) (also referred to as HB C16K), or functional variants, analogsor derivatives thereof.

Additionally, the HB portion of the composition may be repeated,optionally with a linker peptide connecting the HB peptides. Thus, forexample, using SEQ ID NO: 2 as an exemplary HB portion, a therapeuticmolecule can comprise or be linked to the following amino acids:KRKKKGKGLGKKRDPRLRKYKGGGKRKKKGKGLGKKRDPRLRKYK (SEQ ID NO: 4) orKRKKKGKGLGKKRDPRLRKYKGGGKRKKKGKGLGKKRDPRLRKYKGGGKRKKKGK GLGKK RDPRLRKYK(SEQ ID NO: 5). Linker peptides commonly known to one of ordinary skillin the art are encompassed for use in the present invention, forexample, such as those as disclosed herein. In some embodiments, alinker peptide comprises GGG. In some embodiments, a linker peptidecomprises (GGGGS) (SEQ ID NO: 42).

In certain aspects, compounds and methods of the invention may relate tomodifying the pharmacokinetic properties, including the retention intissue, the release to systemic circulation, and the systemic half-lifeof a therapeutic moiety by incorporating one or more of the HB domainsdescribed above. For example, the inventors have shown that a C17Smodified HB-IGF-1 fusion protein remains detectable in skin tissue forat least 24 hours after intradermal injection where IGF-1 alone wasundetectable after 6 hours. In another example, the inventors found thatmore than three times the amount of C17S modified HB-IGF-1 fusionprotein was retained in skin tissue than IGF-1 alone 24 hours aftersubcutaneous injection. Accordingly, HB fused therapeutic moieties ofthe invention, such as C17S modified HB-IGF-1, may be released intosystemic circulation at a slower rate than therapeutic moieties alone,such as IGF-1.

In certain aspects, the invention provides a composition for controlledsystemic release of a therapeutic. The composition includes atherapeutic moiety and at least one heparin binding peptide linked tothe therapeutic moiety, wherein the heparin binding peptide includes aportion with a sequence differing from KRKKKGKGLGKKRDPCLRKYK (SEQ IDNO: 1) by at least one amino acid and that preferably shares at least80% amino acid identity with KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1). In apreferred embodiment, the sequence shares at least 90% or 95% amino acididentity with KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1). In some embodiments,the sequence is KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1) except that the Cat the 16^(th) position has been replaced by the at least one aminoacid. In certain embodiments, the sequence is KRKKKGKGLGKKRDPRLRKYK (SEQID NO: 2) (C16R), KRKKKGKGLGKKRDPKLRKYK (SEQ ID NO: 3) (C16K), orKRKKKGKGLGKKRDPSLRKYK (SEQ ID NO: 82) (C16S). The composition mayinclude a second (optional third, fourth, etc.) heparin binding peptidesharing at least 80% amino acid identity with KRKKKGKGLGKKRDPCLRKYK (SEQID NO: 1).

In some embodiments, the therapeutic moiety comprises insulin-likegrowth factor 1 (IGF-1) or Interleukin 1 receptor antagonist (IL-IRA).The composition may also include any of a polyhistidine tag, a FLAG-tag,or a hydrogel. Amino acid sequences (e.g., HB domains linked toproteins) may be produced by expression in E. coli. The therapeuticmoiety may include a Neurotrophic factor. Such factors may be, forexample, neurotrophins, glial cell-line derived neurotrophic factorfamily ligands, and neuropoietic cytokines. Therapeutic moiety mayinclude a therapeutic antibody or portion thereof.

Aspects of the invention provide a composition for increasing retentionof a therapeutic in tissue. The composition includes a therapeuticmoiety and at least one heparin binding peptide linked to thetherapeutic moiety, wherein the heparin binding peptide includes aportion with a sequence differing from KRKKKGKGLGKKRDPCLRKYK (SEQ IDNO: 1) by at least one amino acid and that preferably shares at least80% amino acid identity with KRKKKGKGLGKKRDPCLRKYK (SEO ID NO: 1). In apreferred embodiment, the sequence shares at least 90% or 95% amino acididentity with KRKKKGKGLGKKRDPCLRKYK (SEO ID NO: 1). In some embodiments,the sequence is KRKKKGKGLGKKRDPCLRKYK (SEO ID NO: 1) except that the Cat the 16^(th) position has been replaced by the at least one aminoacid. In certain embodiments, the sequence is KRKKKGKGLGKKRDPRLRKYK (SEQID NO: 2) (C16R), KRKKKGKGLGKKRDPKLRKYK (SEQ ID NO: 3) (C16K), orKRKKKGKGLGKKRDPSLRKYK (SEQ ID NO: 82) (C16S). The composition mayinclude a second (optional third, fourth, etc.) heparin binding peptidesharing at least 80% amino acid identity with KRKKKGKGLGKKRDPCLRKYK (SEQID NO: 1).

In some embodiments, the therapeutic moiety comprises an immunogenicentity. The immunogenic entity may be one that elicits an immuneresponse in the patient to a disease such as anthrax, measles, rubella,cholera, meningococcal disease, influenza, diphtheria, mumps, tetanus,hepatitis A, pertussis, tuberculosis, hepatitis B, pneumococcal disease,typhoid fever, hepatitis E, poliomyelitis, tick-born encephalitis,haemophilus influenzae type b, rabies, varicella and herpes zoster(shingles), human papilloma-virus, rotavirus gastroenteritis, yellowfever, or Japanese encephalitis.

In certain embodiments, the therapeutic moiety comprises an allergenicentity. The allergenic entity may be one that induces tolerance in thepatient of an allergen such as pollen, pet dander, dust mites, airbornemolds, tree nuts, peanuts, fruits, milk, eggs, fish, shellfish, honeybee venom, yellow jacket venom, hornet venom, wasp venom, or fire antvenom.

In some aspects, the invention provides a method of treating a patient.The method includes administering a compound to a location within theskin of a patient. The compound comprises at least one heparin bindingpeptide and a therapeutic moiety. The heparin binding peptide modifiesthe pharmacokinetic properties of the therapeutic moiety within skinrelative to the therapeutic moiety alone. The compound may include aplurality of heparin binding peptides to elicit a desired releaseprofile for the compound. The compound may be administered by, forexample, intradermal injection, subcutaneous injection, epidermaldeliver, or transdermal delivery. Therapeutic moiety comprises animmunogenic entity, an allergenic entity, a Neurotrophic factor, atherapeutic antibody or portion thereof, an insulin-like growth factor 1(IGF-1), an Interleukin 1 receptor antagonist (IL-IRA), others, orcombinations thereof. The compound may include a hydrogel.

In certain aspects, the invention provides a composition that includes atherapeutic moiety and at least one heparin binding peptide linked tothe therapeutic moiety, wherein the heparin binding peptide includes aportion with a sequence differing from KRKKKGKGLGKKRDPCLRKYK (SEQ IDNO: 1) by at least one amino acid and that preferably shares at least80% amino acid identity with KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1). In apreferred embodiment, the sequence shares at least 90% or 95% amino acididentity with KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1). In some embodiments,the sequence is KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1) except that the Cat the 16^(th) position has been replaced by the at least one aminoacid. In certain embodiments, the sequence is KRKKKGKGLGKKRDPRLRKYK (SEQID NO: 2), KRKKKGKGLGKKRDPKLRKYK (SEQ ID NO: 3), orKRKKKGKGLGKKRDPSLRKYK (SEQ ID NO: 82). The composition may include asecond (optional third, fourth, etc.) heparin binding peptide sharing atleast 80% amino acid identity with KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1).The composition may include a plurality of heparin binding peptides toelicit a desired release profile for the compound. The composition maybe delivered by intradermal injection, subcutaneous injection, ortransdermal delivery.

The therapeutic moiety can be an immunomodulator such as an agent thatstimulates or inhibits an immune response, e.g., a vaccine or allergen,respectively. The agent may stimulate an immune response to a diseasesuch as cancer, anthrax, measles, rubella, cholera, meningococcaldisease, influenza, diphtheria, mumps, tetanus, hepatitis A, pertussis,tuberculosis, hepatitis B, pneumococcal disease, typhoid fever,hepatitis E, poliomyelitis, tick-born encephalitis, haemophilusinfluenzae type b, rabies, varicella and herpes zoster (shingles), humanpapilloma-virus, rotavirus gastroenteritis, yellow fever, or Japaneseencephalitis. The agent may induce tolerance to pollen, pet dander, dustmites, airborne molds, tree nuts, peanuts, fruits, milk, eggs, fish,shellfish, honey bee venom, yellow jacket venom, hornet venom, waspvenom, or fire ant venom.

An immunomodulator can induce, stimulate, enhance or suppress an immuneresponse. An immunomodulator that stimulates (e.g., elicits oramplifies) an immune response are often referred to as activationimmunotherapies and can be used, for example, to induce an immuneresponse to an antigen, e.g., by inducing an antibody response and/or aT-cell mediated immune response. Such immunomodulators that stimulate animmune response can be used to elicit an immune response to a pathogenicantigen, or a cancer antigen, or for autologous immune enhancementtherapy (AIET). An immunomodulator that can inhibit (or suppress) animmune response are often referred to as suppression immunotherapies,and can be used, e.g., to dampens or suppress an abnormal immuneresponse to an allergen, or in autoimmune diseases or reduces a normalimmune response to prevent rejection of transplanted organs or cells.

In some embodiments, a therapeutic moiety may be a Neurotrophic factorsuch as a neurotrophin, a glial cell-line derived neurotrophic factorfamily ligand, or a neuropoietic cytokine. The therapeutic moiety may bea therapeutic antibody or portion thereof. The therapeutic moiety mayinclude insulin-like growth factor 1 (IGF-1), Interleukin 1 receptorantagonist (IL-IRA), a hemophiliac inhibitor, factor VIII (fVIII), a Trkkinase inhibitor, C Natriuretic peptide, Kartigenin, or Trofinetide.

Compounds or compositions of the present invention provide for treatinga neurological condition (e.g., a disorder or disease). Related methodsinclude administering to a subject an effective amount of a recombinantfusion protein comprising HB-X, where X is a therapeutic protein or aportion thereof, selected from nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4(NT-4), Ciliary neurotrophic factor (CNTF), mesencephalicastrocyte-derived neurotrophic factor (MANF), conserved dopamineneurotrophic factor (CDNF), glial cell line-derived neurotrophic factor(GDNF), neurturin (NRTN), artemin (ARTN), persephin (PSPN),interleukin-6, interleukin-11, interleukin-27, leukaemia inhibitoryfactor, ciliary neurotrophic factor, cardiotrophin 1, neuropoietin,cardiotrophin-like cytokine, FPF-1070, Fibroblast Growth Factor 2,Neuregulin-1, Vascular endothelial growth factor (VEGF), IGF orInsulin-like Growth Factor 1 (IGF-1).

The neurological condition can be selected from Alzheimer's disease,Parkinson's disease, Amyotrophic lateral sclerosis, Multiple sclerosis,Brain injury, Spinal cord injury, Peripheral nerve degeneration, Stroke,Huntington's disease, Pick's disease, Diabetic neuropathy,Frontotemporal dementia, Dementia with Lewy bodies, Corticobasaldegeneration, Progressive supranuclear palsy, Prion disorders,Progressive supranuclear palsy, Multiple system atrophy, Hereditaryspastic paraparesis, Spinocerebellar atrophies, Friedreich's ataxia,Amyloidoses, or Charcot Marie Tooth syndrome.

Another embodiment provides for the administration of a HB-X compositionfor the treatment of eye diseases such as Corneal ulcer, Cornealabrasion, Thygeson's superficial punctate keratopathy, Cornealneovascularization, Fuchs' dystrophy, Keratoconjunctivitis sicca,Chorioretinal inflammation, Chorioretinal scars, Choroidal degeneration,Hereditary choroidal dystrophy, Retinal detachment, Retinoschisis,Hypertensive retinopathy, Retinopathy of prematurity, Age-relatedmacular degeneration, Retinal degeneration, Macular degeneration,Epiretinal membrane, Peripheral retinal degeneration, Hereditary retinaldystrophy, Retinitis pigmentosa, Xerophthalmia, or Retinal haemorrhage.

Another aspect of the present invention provides for a method oftreating inflammation comprising administering to a subject an effectiveamount of a recombinant fusion protein comprising HB-X, where X is atherapeutic protein or a portion thereof, selected from TNF receptor 2,interleukin-4, or interleukin-10.

In another embodiment of the invention, the HB-X is provided in asustained release vehicle, such as hyaluronic acid, to further augmentthe release and physiological effect of the HB-X composition.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides data showing long term retention of an embodiment ofHB-IGF-1 after intraarticular injection. Western blot analysis wasperformed for retained IGF-1 or HB-IGF-1 in rat articular cartilage,meniscus, or patellar tendon at 2, 4, 6, and 8 days afterintra-articular injection of either IGF-1, HB-IGF-1, or PBS.

FIG. 2 shows serum levels of IGF-1 vs. HB-IGF-1 after intra-articularinjection. Male Lewis rats 251-275 g (Charles River, Wilmington Mass.)were randomly assigned to one of three groups (n=3 for each group)HB-IGF-1, IGF-1 or Saline. Rats received 50 μm intra-articularinjections containing either 100 μg of HB-IGF-1, 100 μg IGF-1, or Salinein the right knee joint. Blood was harvested via tail vein at 2, 4, 8,24, 48, and 96 hours after injection. Serum levels were measured with anELISA (R&D Systems, Minneapolis, Minn.). HB-IGF-1 levels in serum weresignificantly lower than IGF-1 levels at the first three time points.HB-IGF-1 levels were not significantly different from Saline after 2hours. This shows that intra-articular injection of HB-IGF-1 limits theamount of non-specific IGF-1 circulation compared with non-HB associatedIGF-1.

FIG. 3 is a bar graph depicting ex vivo sustained stimulation ofcartilage biosynthesis and proliferation by HB-IGF-1 afterintra-articular injection in rats. Rats received a single 50 μgintra-articular injection containing either 100 m of an embodiment ofHB-IGF-1, 100 μg IGF-1, or PBS in the right knee joint. Rats weresacrificed 2 and 4 days after the injection and the meniscus washarvested and cultured with radiolabel. Graph represents [35S]sulfateincorporation in the meniscus 2 and 4 days after intraarticularinjection. Results are shown as mean±SEM.

FIG. 4 is a bar graph from an osteoarthritis study comparing OARSIscores of HB-IGF 1, IGF-1, and PBS.

FIG. 5 shows superior expression of soluble HB-IGF-1 comprising enhancedHB (eHB) peptides: C17K (SEQ ID NO: 22) and C17R (SEQ ID NO: 21). FIG. 5shows a Western blot using an anti-IGF-1 antibody to detect the presenceof HB-IGF-1 after expression in E. coli of different HB-IGF-1 fusionvariants, where the HB variants include C17K (SEQ ID NO: 22), C17R (SEQID NO: 21), C17S (SEQ ID NO: 41), and wild-type HB (SEQ ID NO: 20). FIG.5 shows that HB-IGF-1 fusion proteins comprising C17K (SEQ ID NO: 22) orC17R (SEQ ID NO: 21) variants result in greater production of solubleHB-IGF-1 protein as compared to the C17S (SEQ ID NO: 41) and wild-type(SEQ ID NO: 1) HB variants.

FIG. 6 shows superior yield on purification of soluble HB-IGF-1 with anHB-IGF-1 fusion protein comprising the C17R (SEQ ID NO: 21) HB variant.FIG. 6 shows a Western blot using an anti-IGF-1 antibody to detectHB-IGF-1 purified by size exclusion chromatography after production inE. coli of HB-IGF-1 fusion variants comprising either the C17R (SEQ IDNO: 21) or wild-type (SEQ ID NO: 20) HB peptide. FIG. 6 shows asignificantly higher yield of E. coli-expressed soluble HB-IGF-1 afterpurification for HB(C17R)—IGF-1 fusion proteins as compared toHB(WT)-IGF-1 fusion proteins.

FIG. 7 shows HB (C17R) allows superior yield of HB-IGF-1 from E. coliinclusion bodies. FIG. 7 shows a Western blot using an anti-IGF-1antibody to detect HB-IGF-1 protein in extracts purified from inclusionbodies after expression in E. coli of HB-IGF1 fusion variants comprisingeither the C17R (SEQ ID NO: 21) (referred to as eHB) or wild-type (SEQID NO: 20) HB peptide. FIG. 7 shows a significantly higher yield ofHB-IGF-1 extracted from inclusion bodies in cells expressingHB(C17R)-IGF-1 (eHB-IGF-1) fusion proteins as compared to extracts fromcells expressing HB(WT)-IGF-1 (wHB-IGF-1) fusion proteins.

FIG. 8 shows a western blot of the retention of IGF-1 in the spinal cordincubated with HB-IGF-1. Rat spinal cord tissue was incubated for 24hours in medium with no additions (No IGF-1), 1 ug/ml IGF-1, or 1 ug/mlHB-IGF-1. After incubation, tissue was washed and either frozenimmediately (“Day 0”) or incubated for an additional 24 hours in freshmedium before collection to wash out the proteins (“Day 1”). Tissueswere then analyzed by Western blotting with an anti-IGF1 antibody todetect the presence of IGF-1 or HB-IGF-1 remaining in the spinal cordtissue.

FIGS. 9A-9B show that retention of PTH in cartilage disks incubated withPTH-HB. Cartilage disks were incubated in medium with no added peptide(“No PTH”), parathyroid hormone 1-34 (“PTH”), or a fusion of parathyroidhormone 1-34 with an HB domain (“PTH-HB”). FIG. 9A shows the peptidecontrol of amount of PTH or PTH-HB added to the cartilage disk explants.After 24 hours, cartilage discs were washed and returned to incubationin fresh medium in the absence of a peptide. FIG. 9B shows a westernblot with an anti-PTH antibody in cartilage tissue extracts two daysafter washout, showing that PTH is only retained in the cartilage discsincubated with PTH-HB but not non-fused PTH.

FIG. 10 shows extended retention of an embodiment of HB-IGF-1 afterintradermal injection. Western blot analysis was performed for retainedIGF-1 or HB-IGF-1 in rat skin at 6 hours, 12 hours, and 24 hours afterintradermal injection of an equal amount of either IGF-1 or HB-IGF-1.

FIG. 11 shows a bar graph depicting higher in vivo retention in skin ofa 14C labeled embodiment of HB-IGF-1 in tissue 24 hours aftersubcutaneous injection. Rats received a 100 μg subcutaneous injection ofeither 14C-HB-IGF-1 (C17S) or 14C-IGF-1. Tissue was collected 24 hoursafter injection, digested and radioactivity was measured byscintillation counting.

DETAILED DESCRIPTION

The present invention is related to conjugation of one or moreheparin-binding peptides (HB) to an active agent (e.g., X) forcontrolled delivery of the active agent. Heparin-binding domains maybind to negatively charged proteoglycans in cartilage, brain and spinalcord tissue, as well as skin and subcutaneous tissues. Accordingly,therapeutic HB fusions of the invention may be designed to change thepharmacokinetics of therapies after subcutaneous or intradermaldelivery. For example, an HB domain fused to a therapeutic protein orpeptide can extend release of the therapy into the systemic circulationafter injection, thus allowing the therapy to be delivered by lessfrequent injections. In another example, an immunogenic entity in avaccine may be fused to an HB domain in order to increase its retentionafter intradermal injection and thus provide a stronger immune responseto the vaccine. As a third example, fusion of an allergen to an HBdomain may be used to enhance induction of tolerance in a patientthrough allergen immunotherapy by prolonging the residence time of theallergen in the skin and thus increasing its ability to interact withcells in the dermis.

Certain embodiments herein are directed to heparin-binding peptides (HB)of SEQ ID NO: 1-3, 20-22, 41, or 82 fused to an active agent which is atherapeutic protein or a portion thereof, optionally by a linkerpeptide. Some embodiments, the active agent is a small molecule, andenable delivery of the small molecule to cells and tissues comprisingproteoglycans, where a HB peptide of SEQ ID NO: 1-3, 20-22, 41, or 82can be conjugated to the small molecule by direct or indirect linkage(e.g., use of chemical linkers). In some embodiments, compositionscomprising HB-X conjugates can be used for delivery of one or multipledifferent therapeutic proteins and agents to tissues an cells expressingproteoglycans.

Heparin-binding peptides or domains, as contemplated by the invention,may share, for example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% amino acid identity with SEQ ID NO: 1. In certainaspects, a heparin-binding domain may be modified so that a cysteineresidue of the heparin binding peptide has been replaced with adifferent amino acid residue, including, for example the 16th residue inSEQ ID NO: 1, which may be replaced with, for example, an Arginine,Lysine, or Serine residue.

An aspect of the present invention provides for a selective therapeuticcomposition comprising HB-Xn, or (HB-linker)n-Xn, where HB is a heparinbinding protein, X is an active agents such as a therapeutic protein ora portion thereof, or a therapeutic small molecule, and n is an integerof at least 1. In some embodiments, the composition is a recombinantfusion protein comprising a recombinant HB and the therapeutic protein(or portion thereof). Any combination of a HB peptide selected from thegroup of SEQ ID NO: 1-3, 20-22, 41, or 82 can be used in any combinationof an active agent, with or without the presence of a linker protein,where the HB peptide can be located at the N- and/or C-terminus of theactive agent, and there can be one or multiple HB peptide-linkersattached to the N- and/or C-terminus of the active agent. For example,in some embodiments, the fusion or conjugate can comprise(HB-linker)n-Xm-(linker-HB)0, where n, m, and o are integers, and m isat least one and n+o is at least one.

In some embodiments, the components of the composition can be placed inorder, relative to the N-terminus of the HB portion of the composition:HB-X, X-HB, HB-linker-X, (HB-linker)2-X, X-linker-HB, X-HB-X, HBn-X-HBn,(HB-linker)n-X-(linker-HB)n, HB-X-HB-X, etc. Additionally, thecomposition can comprise a mixture of HB-X constructs, wherein Xrepresents different proteins or small molecules (i.e., a compositioncomprising HB-X1 and HB-X2, etc.). An example linker is a peptide havingthe amino acids GGG.

In certain embodiments, compounds of the invention may include a proteintag such as a FLAG tag, amino acid sequence DYKDDDDK (SEQ ID NO: 84),for detection of the compound or a polyhistidine-tag (e.g., a 6×His tag,amino acid sequence HHHHHH (SEQ ID NO: 85)) for isolation of thecompound. Protein tags may be incorporated at any point in the compound.Protein tags may be linked to the compound by cleavable linkers,allowing protein tags used in expression to be cleaved beforeadministration of the compound to a patient. In an exemplary embodiment,protein tags are positioned at the C-terminus of the compound, producinga composition such as HB-linker-X-linker-tag-linker-tag.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. All Gene IDs refer tohuman genes, unless otherwise noted, available in the National Centerfor Biotechnology Information (NCBI) database.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.The term “or” is inclusive unless modified, for example, by “either.”Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

The present invention provides for controlled release of active agentsand therapeutic moieties, e.g. proteins or small molecules, toparticular tissues to which heparin binding proteins associate and,accordingly, controlled systemic release of those agents from thetissues. More specifically, the present embodiments provide for novelproteinaceous heparin-binding motifs (HB) that are linked to or fused toa therapeutic moiety, such as a small molecule, cytokine, growth factor,allergenic entity, immunogenic entity, or functional portion thereof.

Definitions

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

The term “protein” may be used interchangeably with “polypeptide” torefer to a polymer of amino acid residues linked by peptide bonds.Typically, a protein or polypeptide has a minimum length of at least 25amino acids. The term “polypeptide” and “protein” can encompass amultimeric protein, e.g., a protein containing more than one domain orsubunit. The term “peptide” as used herein typically refers to a peptidebond-linked amino acid polymer containing less than 25 amino acids,e.g., between about 4 amino acids and about 25 amino acids in length.Proteins and peptides can be composed of linearly arranged amino acidslinked by peptide bonds, whether produced biologically, recombinantly,or synthetically and whether composed of naturally occurring ornon-naturally occurring amino acids, are included within thisdefinition. Both full-length proteins and fragments thereof greater than25 amino acids are encompassed by the definition of protein. The termsalso include polypeptides that have co-translational (e.g., signalpeptide cleavage) and post-translational modifications of thepolypeptide, such as, for example, disulfide-bond formation,glycosylation, acetylation, phosphorylation, lipidation, proteolyticcleavage (e.g., cleavage by metalloproteases), and the like.Furthermore, as used herein, a “polypeptide” refers to a protein thatincludes modifications, such as deletions, additions, and substitutions(generally conservative in nature as would be known to a person in theart) to the native sequence, as long as the protein maintains thedesired activity. These modifications can be deliberate, as throughsite-directed mutagenesis, or can be accidental, such as throughmutations of hosts that produce the proteins, or errors due to PCRamplification or other recombinant DNA methods. Polypeptides or proteinsare composed of linearly arranged amino acids linked by peptide bonds,but in contrast to peptides, has a well-defined conformation. Proteins,as opposed to peptides, generally consist of chains of 25 or more aminoacids. For the purposes of the present invention, the term “peptide” asused herein typically refers to a sequence of amino acids of made up ofa single chain of D- or L-amino acids or a mixture of D- and L-aminoacids joined by peptide bonds. Generally, peptides contain at least twoamino acid residues and are less than about 25 amino acids in length.

It will be appreciated that proteins, polypeptides, or peptides oftencontain amino acids other than the 20 amino acids commonly referred toas the 20 naturally occurring amino acids (e.g., synthetic non-nativeamino acids), and that many amino acids, including the terminal aminoacids, can be modified in a given polypeptide, either by naturalprocesses such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Known modifications which can be present inpolypeptides of the present invention include, but are not limited to,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa polynucleotide or polynucleotide derivative, covalent attachment of alipid or lipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formulation, gamma-carboxylation, glycation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination.

The incorporation of non-natural amino acids, including syntheticnon-native amino acids, substituted amino acids, or one or more D-aminoacids into the HB peptides and/or active agent peptides or proteins (orother components of the composition) is desirable in certain situations.D-amino acid-containing peptides exhibit increased stability in vitro orin vivo compared to L-amino acid-containing forms. Thus, theconstruction of peptides incorporating D-amino acids can be particularlyuseful when greater in vivo or intracellular stability is desired orrequired. More specifically, D-peptides are resistant to endogenouspeptidases and proteases, thereby providing better oral trans-epithelialand transdermal delivery of linked drugs and conjugates, improvedbioavailability of membrane-permanent complexes (see below for furtherdiscussion), and prolonged intravascular and interstitial lifetimes whensuch properties are desirable. The use of D-isomer peptides can alsoenhance transdermal and oral trans-epithelial delivery of linked drugsand other cargo molecules. Additionally, D-peptides cannot be processedefficiently for major histocompatibility complex class II-restrictedpresentation to T helper cells, and are therefore less likely to inducehumoral immune responses in the whole organism. Peptide conjugates cantherefore be constructed using, for example, D-isomer forms of cellpenetrating peptide sequences, L-isomer forms of cleavage sites, andD-isomer forms of therapeutic peptides. In some embodiments, a HB-fusionprotein comprises D- and/or L-amino acid residues, as use of naturallyoccurring L-amino acid residues has the advantage that any break-downproducts should be relatively non-toxic to the cell or organism.

In yet a further embodiment, HB-X can be a retro-inverso peptides. A“retro-inverso peptide” refers to a peptide with a reversal of thedirection of the peptide bond on at least one position, i.e., a reversalof the amino- and carboxy-termini with respect to the side chain of theamino acid. Thus, a retro-inverso analogue has reversed termini andreversed direction of peptide bonds while approximately maintaining thetopology of the side chains as in the native peptide sequence. Theretro-inverso peptide can contain L-amino acids or D-amino acids, or amixture of L-amino acids and D-amino acids, up to all of the amino acidsbeing the D-isomer. Partial retro-inverso peptide analogues arepolypeptides in which only part of the sequence is reversed and replacedwith enantiomeric amino acid residues. Since the retro-inverted portionof such an analogue has reversed amino and carboxyl termini, the aminoacid residues flanking the retro-inverted portion are replaced byside-chain-analogous a-substituted geminal-diaminomethanes andmalonates, respectively. Retro-inverso forms of cell penetratingpeptides have been found to work as efficiently in translocating acrossa membrane as the natural forms. Synthesis of retro-inverso peptideanalogues are described in Bonelli, F. et al., Int J Pept Protein Res.24(6):553-6 (1984); Verdini, A. and Viscomi, G. C, J. Chem. Soc. PerkinTrans. 1:697-701 (1985); and U.S. Pat. No. 6,261,569, which areincorporated herein in their entirety by reference. Processes for thesolid-phase synthesis of partial retro-inverso peptide analogues havebeen described (EP 97994-B) which is also incorporated herein in itsentirety by reference.

The term “variant” refers to a polypeptide or nucleic acid that differsfrom the naturally occurring polypeptide or nucleic acid by one or moreamino acid or nucleic acid deletions, additions, substitutions orside-chain modifications, yet retains one or more specific functions orbiological activities of the naturally occurring molecule. Amino acidsubstitutions include alterations in which an amino acid is replacedwith a different naturally-occurring or a non-conventional amino acidresidue. Such substitutions may be classified as “conservative,” inwhich case an amino acid residue contained in a polypeptide is replacedwith another naturally occurring amino acid of similar character eitherin relation to polarity, side chain functionality or size. Substitutionsencompassed by variants as described herein may also be“non-conservative,” in which an amino acid residue which is present in apeptide is substituted with an amino acid having different properties(e.g., substituting a charged or hydrophobic amino acid with alanine),or alternatively, in which a naturally-occurring amino acid issubstituted with a non-conventional amino acid. Also encompassed withinthe term “variant,” when used with reference to a polynucleotide orpolypeptide, are variations in primary, secondary, or tertiarystructure, as compared to a reference polynucleotide or polypeptide,respectively (e.g., as compared to a wild-type polynucleotide orpolypeptide).

Variants can include conservative or non-conservative amino acidchanges, as described below. Polynucleotide changes can result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. Variants can also includeinsertions, deletions or substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of ornithine which donot normally occur in human proteins. “Conservative amino acidsubstitutions” result from replacing one amino acid with another thathas similar structural and/or chemical properties. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. For example, the following six groups each containamino acids that are conservative substitutions for one another: (1)Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid (D), Glutamicacid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine(K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and(6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See, e.g.,Creighton, PROTEINS (W.H. Freeman & Co., 1984). The choice ofconservative amino acids may be selected based on the location of theamino acid to be substituted in the peptide, for example if the aminoacid is on the exterior of the peptide and exposed to solvents, or onthe interior and not exposed to solvents. In some embodiments,polypeptides including non-conservative amino acid substitutions arealso encompassed within the term “variants.” As used herein, the term“non-conservative” substitution refers to substituting an amino acidresidue for a different amino acid residue that has different chemicalproperties. Non-limiting examples of non-conservative substitutionsinclude aspartic acid (D) being replaced with glycine (G); asparagine(N) being replaced with lysine (K); and alanine (A) being replaced witharginine (R). Selection of such conservative and non-conservative aminoacid substitutions is within the skill of one of ordinary skill in theart.

The term “derivative” refers to proteins or peptides which have beenchemically modified, for example by ubiquitination, labeling, pegylation(derivatization with polyethylene glycol) or addition of othermolecules. A molecule is also a “derivative” of another molecule when itcontains additional chemical moieties not normally a part of themolecule. Such moieties can improve the molecule's solubility,absorption, biological half-life, etc. The moieties can alternativelydecrease the toxicity of the molecule, or eliminate or attenuate anundesirable side effect of the molecule, etc. Moieties capable ofmediating such effects are disclosed in REMINGTON'S PHARMACEUTICALSCIENCES (21st ed., Tory, ed., Lippincott Williams & Wilkins, Baltimore,Md., 2006).

The term “functional” when used in conjunction with “derivative” or“variant” refers to a protein molecule which possesses a biologicalactivity that is substantially similar to a biological activity of theentity or molecule of which it is a derivative or variant.“Substantially similar” in this context means that the biologicalactivity of a polypeptide, is at least 50% as active as a reference,e.g., a corresponding wild-type polypeptide, e.g., at least 60% asactive, 70% as active, 80% as active, 90% as active, 95% as active,100%) as active or even higher (i.e., the variant or derivative hasgreater activity than the wild-type), e.g., 110%> as active, 120%) asactive, or more, inclusive.

The term “functional portion” or “functional fragment” refers to aportion of the native molecule (e.g., the native protein or receptorbinding moiety of a chemical entity) that mediates the same effect asthe full-length molecule, e.g., stimulates a cell response such asgrowth or affects a signal or signal cascade related to a desiredphysiological effect.

The term “fragment” of a peptide, polypeptide or molecule as used hereinrefers to any contiguous polypeptide subset of the molecule. The term“protein fragment” as used herein includes both synthetic andnaturally-occurring amino acid sequences derivable from the naturallyoccurring amino acid sequence, e.g., a naturally occurring active agentwhich is a protein, or HB (SEQ ID NO: 1) or a variant thereof (e.g., SEQID NO: 2 or SEQ ID NO: 3). The protein is said to be “derivable from thenaturally-occurring amino acid sequence” if it can be obtained byfragmenting the naturally-occurring protein, or if it can be synthesizedbased upon a knowledge of the sequence of the naturally occurring aminoacid sequence or of the genetic material (DNA or RNA) which encodes thissequence.

Accordingly, a “fragment” of a molecule, is meant to refer to anypolypeptide subset of the molecule. Fragments of HB which have theactivity of HB peptide variants of SEQ ID NO: 2 or SEQ ID NO: 3 asdisclosed herein and which are soluble are also encompassed for use inthe present invention.

For example functional fragments of SEQ ID NO: 2 or SEQ ID NO: 3 usefulin the methods as disclosed herein have at least 30% the activity asthat of a polypeptide of SEQ ID NO: 2 or SEQ NO: 3 in vivo. Statedanother way, a fragment of SEQ ID NO: 2 or SEQ ID NO: 3 is any fragmentwhich, alone or fused to an active agent can result in at least 30% ofthe same activity as compared to SEQ ID NO: 2 or SEQ ID NO: 3 to retainthe HB-fusion protein in the tissue after 24 hours after wash-out asdisclosed herein when a HB fusion protein comprising SEQ ID NO: 2 or SEQID NO: 3 is incubated with a cartilage explant or spinal cord explant(as disclosed in the Examples). A “fragment” can be at least about 6, atleast about 9, at least about 15, at least about 20, at least about 30,least about 40, at least about 50, at least about 100, at least about250, at least about 300 nucleic or amino acids, and all integers inbetween. Exemplary fragments include C-terminal truncations, N-terminaltruncations, or truncations of both C- and N-terminals (e.g., deletionsof, for example, at least 1, at least 2, at least 3, at least 4, atleast 5, at least 8, at least 10, at least 15, at least 20, at least 25,at least 40, at least 50, at least 75, at least 100 or more amino acidsdeleted from the N-termini, the C-termini, or both). One of ordinaryskill in the art can create such fragments by simple deletion analysis.Such a fragment of SEQ ID NO: 2 or SEQ ID NO: 3 can be, for example, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or more than 10 amino acids,deleted from the N-terminal and/or C-terminal of SEQ ID NO: 2 or SEQ IDNO: 3, respectively. In some embodiments, by sequentially deleting N-and/or C-terminal amino acids from SEQ ID NO: 2 or SEQ ID NO: 3, andassessing the function of the resulting peptide fragment, alone or fusedto an active agent can identify a functional fragment of HB for use inthe present invention. One can create functional fragments with multiplesmaller fragments. These can be attached by bridging peptide linkers.One can readily select linkers to maintain wild type conformation. Oneof ordinary skill in the art can easily assess the function of an HB-Xconjugate to retain in the tissue and cause a biological effect by theactive agent X (as disclosed in the Examples) as compared to a HB-fusionprotein comprising SEQ ID NO: 2 or SEQ ID NO: 3. Using an in vivo assaysuch as the cartilage assay as disclosed in the Examples, if the HBpeptide fragment has at least 30% of the biological activity of the HBcorresponding to SEQ ID NO: 2 or SEQ ID NO: 3 as disclosed herein, thenthe HB peptide fragment of an HB(fragment)-X fusion protein isconsidered a valid HB-fragment and can used in fusion proteins andmethods as disclosed herein. In some embodiments, a fragment of SEQ IDNO: 2 or SEQ ID NO: 3 can be less than 20, or less than 15 or less than10, or less than 5 amino acids of SEQ ID NO: 2 or SEQ ID NO: 3. However,as stated above, the fragment must be at least 4 amino acids, at leastabout 9, at least about 15, at least about 20, at least about 30, atleast about 40, at least about 50, at least about 100, at least about250, at least about 500 nucleic acids or amino acids, or any integers inbetween.

The term “wild type” refers to the naturally-occurring, normalpolynucleotide sequence encoding a protein, or a portion thereof, orprotein sequence, or portion thereof, respectively, as it normallyexists in vivo.

The term “mutant” refers to an organism or cell with any change in itsgenetic material, in particular a change (i.e., deletion, substitution,addition, or alteration) relative to a wild-type polynucleotide sequenceor any change relative to a wild-type protein sequence. The term“variant” may be used interchangeably with “mutant”. Although it isoften assumed that a change in the genetic material results in a changeof the function of the protein, the terms “mutant” and “variant” referto a change in the sequence of a wild-type protein regardless of whetherthat change alters the function of the protein (e.g., increases,decreases, imparts a new function), or whether that change has no effecton the function of the protein (e.g., the mutation or variation issilent).

The term “substantially similar,” when used in reference to a variant ofa protein or peptide or a functional derivative thereof, as comparedwith the original protein, means that a particular subject sequencevaries from the sequence of the polypeptide by one or moresubstitutions, deletions, or additions, but retains at least 50%, orhigher, e.g., at least 60%>, 70%, 80%, 90% or more, inclusive, of thefunction of the protein. In determining polynucleotide sequences, allsubject polynucleotide sequences capable of encoding substantiallysimilar amino acid sequences are considered to be substantially similarto a reference polynucleotide sequence, regardless of differences incodon sequence. A nucleotide sequence is “substantially similar” to agiven nucleic acid sequence if: (a) the given polynucleotide nucleotidehybridizes to the coding regions of the native polynucleotide, or (b)the given polynucleotide is capable of hybridization to the nativepolynucleotide under moderately stringent conditions and its encodedprotein has biological activity similar to the native protein; or (c)the sequence of polynucleotide are degenerate as a result of the geneticcode relative to the nucleotide sequences defined in (a) or (b).Substantially similar proteins will typically be greater than about 80%similar to the corresponding sequence of the native protein.

The terms “homologous” or “homologues” are used interchangeably, andwhen used to describe a polynucleotide or polypeptide, indicate that twopolynucleotides or polypeptides, or designated sequences thereof, whenoptimally aligned and compared, for example using BLAST, version 2.2.14with default parameters for an alignment are identical, with appropriatenucleotide insertions or deletions or amino-acid insertions ordeletions, typically in at least 70% of the nucleotides of thenucleotides for high homology. For a polypeptide, there should be atleast 30% of amino acid identity in the polypeptide, or at least 50% forhigher homology. The term “homolog” or “homologous” as used herein alsorefers to homology with respect to structure. Determination of homologsof genes or polypeptides can be easily ascertained by the skilledartisan. When in the context with a defined percentage, the definedpercentage homology means at least that percentage of amino acidsimilarity. For example, 85% homology refers to at least 85% of aminoacid similarity.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters. Where necessary or desired, optimalalignment of sequences for comparison can be conducted by any variety ofapproaches, as these are well-known in the art.

The term “heterologous” in reference to nucleic acid sequences, proteinsor polypeptides, means that these molecules are not naturally occurringin that cell. For example, the nucleic acid sequence coding for a fusionprotein described herein that is inserted into a cell, e.g. in thecontext of a protein expression vector, is a heterologous nucleic acidsequence.

The term “agent” or “compound” as used herein refers to a chemicalentity or biological product, or combination of chemical entities orbiological products, administered to a subject to treat or prevent orcontrol a disease or condition. The chemical entity or biologicalproduct is preferably, but not necessarily a low molecular weightcompound, but may also be a larger compound, or any organic or inorganicmolecule, including modified and unmodified nucleic acids such asantisense nucleic acids, RNAi, such as siRNA or shRNA, peptides,peptidomimetics, receptors, ligands, and antibodies, aptamers,polypeptides, nucleic acid analogues or variants thereof. For example,an agent can be an oligomer of nucleic acids, amino acids, orcarbohydrates including, but not limited to proteins, peptides,oligonucleotides, ribozymes, DNAzymes, glycoproteins, RNAi agents (e.g.,siRNAs), lipoproteins, aptamers, and modifications and combinationsthereof. In some embodiments, an active agent is a nucleic acid, e.g.,miRNA or a derivative or variant thereof. In some embodiments, an HB-Xconjugate that comprises a nucleic acid agent, e.g., a RNAi or miRNAagent can be joined (e.g., conjugated) to HB peptide by means of alinker moiety can allow the miRNA or RNAi agent to interact with theDNA. In some embodiments, the linker moiety is a reversible moiety,e.g., miRNA or RNAi agent can be released from the HB peptide at thelocation of the target cell or tissue.

As used herein, the term “fused” means that at least one protein orpeptide is physically associated with a second protein or peptide. Insome embodiments, fusion is typically a covalent linkage, however, othertypes of linkages are encompassed in the term “fused” include, forexample, linkage via an electrostatic interaction, or a hydrophobicinteraction and the like. Covalent linkage can encompass linkage as afusion protein or chemically coupled linkage, for example via adisulfide bound formed between two cysteine residues.

As used herein, the term “fusion polypeptide” or “fusion protein” meansa protein created by joining two or more polypeptide sequences together.The fusion polypeptides encompassed in this invention includetranslation products of a chimeric gene construct that joins the DNAsequences encoding the HB peptide or mutants thereof, with the DNAsequence encoding a second polypeptide to form a single open-readingframe. In other words, a “fusion polypeptide” or “fusion protein” is arecombinant protein of two or more proteins which are joined by apeptide bond or via several peptides. The fusion protein may alsocomprise a peptide linker between the HB peptide and the active agent,e.g., a therapeutic peptide or polypeptide of the fusion protein.

In some embodiments, fusion proteins can be produced, for example, by anucleic acid sequence encoding one protein is joined to the nucleic acidencoding another protein such that they constitute a single open-readingframe that can be translated in the cells into a single polypeptideharboring all the intended proteins. The order of arrangement of theproteins can vary. As a non-limiting example, the nucleic acid sequenceencoding the HB peptide is fused in frame to an end, either the 5′ orthe 3′ end, of a gene encoding a first fusion partner (e.g., X), such asa therapeutic protein or peptide. In this manner, on expression of thegene, the HB peptide is functionally expressed and fused to theN-terminal or C-terminal end of X (e.g., the therapeutic peptide orprotein). In certain embodiments, modification of the polypeptide probeis such that the functionality of the HB peptide remains substantiallyunaffected in terms of its biological activity by fusion to the firstfusion partner X, such as a therapeutic peptide. In some embodiments, anucleic acid construct encoding a HB-X fusion protein also has a nucleicacid sequence which encodes a linker, which is located between nucleicacid encoding the HB peptide and the nucleic acid sequence encoding X(e.g., the therapeutic peptide or protein). In some embodiments, theHB-X fusion protein is configures such that the functionality of the HBpeptide or X (e.g., the therapeutic protein or peptide) is notsignificantly compromised by the fusion.

As used herein, the term “associated with” means that one entity is inphysical association or contact with another. Thus, a HB peptide“associated with” an active agent can be either covalently ornon-covalently joining of the HB peptide to the active agent. Theassociation can be mediated by a linker moiety, particularly where theassociation is covalent. The term “association” or “interaction” or“associated with” are used interchangeably herein and as used inreference to the association or interaction of a HB peptide with theactive agent, either by a direct linkage or an indirect linkage. As usedherein, the term “conjugate” or “conjugation” or “linked” as used hereinrefers to the attachment of two or more entities to form one entity. Aconjugate encompasses both peptide-small molecule conjugates as well aspeptide-protein/peptide conjugates. For example, the methods of thepresent invention provide conjugation of a HB peptide joined withanother entity, for example an active agent, e.g., a moiety such as atherapeutic protein/peptide or small molecule. As disclosed herein, theattachment can be by means of linkers, chemical modification, peptidelinkers, chemical linkers, covalent or non-covalent bonds, or proteinfusion or by any means known to one skilled in the art. The joining canbe permanent or reversible. In some embodiments, several linkermolecules (chemical or peptide linkers) can be included in order to takeadvantage of desired properties of each linker and each protein ormolecule in the conjugate. Flexible linkers and linkers that increasethe solubility of the conjugates are contemplated for use alone or withother linkers as disclosed herein. Peptide linkers can be linked byexpressing DNA encoding the linker to one or more proteins in theconjugate. Linkers can be acid cleavable, photocleavable and heatsensitive linkers. Methods for conjugation are well known by personsskilled in the art and are encompassed for use in the present invention.

Alternatively, two or more entities that are joined can be linked byindirect linkage. An indirect linkage includes an association between aHB peptide and an active agent, wherein the HB peptide and the activeagent are attached via a “linker moiety”, e.g., they are not directlylinked. A direct linkage includes any linkage wherein a linker moiety isnot required. In one embodiment, a direct linkage includes a chemical ora physical interaction wherein the two moieties, i.e. the targetingmoiety and binding moiety interact such that they are attracted to eachother. Examples of direct interactions include covalent interactions,non-covalent interactions, hydrophobic/hydrophilic, ionic (e.g.,electrostatic, coulombic attraction, ion-dipole, charge-transfer), Vander Waals, or hydrogen bonding, and chemical bonding, including theformation of a covalent bond. Accordingly, in one embodiment, atargeting moiety, such as an antibody of fragment thereof and thebinding moiety are not linked via a linker, e.g., they are directlylinked. In a further embodiment, a targeting moiety and the bindingmoiety are electrostatically associated with each other.

The term “conjugated” refers to the attachment of at least two entitiesjoined together. The joining of the two entities can be direct (e.g.,via covalent or non-covalent bonds) or indirect (e.g., via linkers etc.)

The term “linker” refers to any means, entity or moiety used to join twoor more entities. For example a HB peptide as disclosed herein can bejoined to an active agent X (e.g., a therapeutic protein or peptide)using a linker moiety. A linker can be a covalent linker or anon-covalent linker. Examples of covalent linkers include covalent bondsor a linker moiety covalently attached to one or more of the proteins tobe linked. The linker can also be a non-covalent bond, e.g. anorganometallic bond through a metal center such as platinum atom. Forcovalent linkages, various functionalities can be used, such as amidegroups, including carbonic acid derivatives, ethers, esters, includingorganic and inorganic esters, amino, urethane, urea and the like. Toprovide for linking, the effector molecule and/or the probe can bemodified by oxidation, hydroxylation, substitution, reduction etc. toprovide a site for coupling. Linkers can be acid cleavable,photocleavable and heat sensitive linkers. Methods for conjugation arewell known by persons skilled in the art and are encompassed for use inthe present invention. In some embodiments, a linker moiety to attach aHB peptide to a nucleic acid is a cyclo-propapyrroloindole cross-linker.Linker moieties include, but are not limited to, chemical linkermoieties, or for example a peptide linker moiety. In some embodiments, alinker between a HB peptide and an active agent or a peptide linker canbe formed by reacting the polymer and a linker selected e.g., from thegroup consisting of p-nitrophenyl chloroformate,carbonyldiimidazole(CDI), N,N′-disuccinimidyl carbonate(DSC),cis-aconitic anhydride, and a mixture of these compounds. It will beappreciated that modification which do not significantly decrease thefunction of the HB peptide as disclosed herein or the active agent(e.g., therapeutic protein or peptide) are preferred.

The term “recombinant” when used to describe a nucleic acid molecule,means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/orsynthetic origin, which, by virtue of its origin or manipulation, is notassociated with all or a portion of the polynucleotide sequences withwhich it is associated in nature. The term recombinant as used withrespect to a peptide, polypeptide, protein, or recombinant fusionprotein, means a polypeptide produced by expression from a recombinantpolynucleotide. The term recombinant as used with respect to a host cellmeans a host cell into which a recombinant polynucleotide has beenintroduced. Recombinant is also used herein to refer to, with referenceto material (e.g., a cell, a nucleic acid, a protein, or a vector) thatthe material has been modified by the introduction of a heterologousmaterial (e.g., a cell, a nucleic acid, a protein, or a vector).

The term “vectors” refers to a nucleic acid molecule capable oftransporting or mediating expression of a heterologous nucleic acid towhich it has been linked to a host cell; a plasmid is a species of thegenus encompassed by the term “vector.” The term “vector” typicallyrefers to a nucleic acid sequence containing an origin of replicationand other entities necessary for replication and/or maintenance in ahost cell. Vectors capable of directing the expression of genes and/ornucleic acid sequence to which they are operatively linked are referredto herein as “expression vectors”. In general, expression vectors ofutility are often in the form of “plasmids” which refer to circulardouble stranded DNA molecules which, in their vector form are not boundto the chromosome, and typically comprise entities for stable ortransient expression or the encoded DNA. Other expression vectors thatcan be used in the methods as disclosed herein include, but are notlimited to plasmids, episomes, bacterial artificial chromosomes, yeastartificial chromosomes, bacteriophages or viral vectors, and suchvectors can integrate into the host's genome or replicate autonomouslyin the particular cell. A vector can be a DNA or RNA vector. Other formsof expression vectors known by those skilled in the art which serve theequivalent functions can also be used, for example self-replicatingextrachromosomal vectors or vectors which integrates into a host genome.Preferred vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked. The terms“subject” and “individual” are used interchangeably herein, and refer toan animal, for example a human, to whom treatment, includingprophylactic treatment, with the pharmaceutical composition according tothe present invention, is provided. The term “subject” as used hereinrefers to human and non-human animals. The term “non-human animals” and“non-human mammals” are used interchangeably herein includes allvertebrates, e.g., mammals, such as non-human primates, (particularlyhigher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig,goat, pig, cat, rabbits, cows, and non-mammals such as chickens,amphibians, reptiles etc. In one embodiment, the subject is human. Inanother embodiment, the subject is an experimental animal or animalsubstitute as a disease model. The term does not denote a particular ageor sex. Thus, adult and newborn subjects, as well as fetuses, whethermale or female, are intended to be covered. Examples of subjects includehumans, dogs, cats, cows, goats, and mice. The term subject is furtherintended to include transgenic species.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, cartilage,neuronal tissue (brain, spinal cord and neurons), muscles, smoothmuscles, skin and organs.

The term “skin” is intended to include at least the epidermis, dermis,and hypodermis tissue layers, including subcutaneous adipose tissue.

The term “disease” or “disorder” is used interchangeably herein, refersto any alternation in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, disposition, affection.

The term “cartilage-related condition” or “cartilage-related clinicalcondition” refers to any defect in the articular cartilage. The termencompasses, but is not limited to, a rupture or detachment of thecartilage, a meniscal defect including a partial or complete tear,damage or a disease effecting the meniscus and/or patella,osteoarthritis (referred to herein as “OA”), including knee, finger,wrist, hip, ankle, elbow, toe, shoulder, and spinal osteoarthritis,traumatic cartilage rupture or detachment, ankylosing spondylitis,capsulitis, psoriatic arthritis, rheumatoid arthritis (RA), systemiclupus erythematosus, juvenile idiopathic arthritis, Chondropathy,Chondrosarcoma, Chondromalacia, Polychondritis, RelapsingPolychondritis, Slipped epiphysis, Osteochondritis Dissecans,Chondrodysplasia, Costochondritis, X-linked hypophosphatemic rickets,Osteochondroma, Chondrosarcoma (malignant), OsteoarthritisSusceptibility (types 1-6), Spondylosis, Osteochondroses, Primarychondrosarcoma, Chondrodysplasia, Tietze syndrome, Dermochondrocornealdystrophy of Francois, Epiphyseal dysplasia, multiple, (types 1-5),Ossified Ear cartilages with Mental deficiency, Muscle Wasting and BonyChanges, Carpotarsal osteochondromatosis, Achondroplasia,Chondrocalcinosis (types 1-2), Genochondromatosis, Chondrodysplasia(disorder of sex development), Chondroma, Achondrogenesis (types 1A, 1B,2, 3, 4, Langer-Saldino Type), Type IIAchondrogenesis-Hypochondrogenesis, Atelosteogenesis, (type 1, 2 andIII), Pyknoachondrogenesis, Pseudoachondroplasia, Osteoarthropathy offingers, familial, Diastrophic dysplasia, Dyschondrosteosis-nephritis,Coloboma of Alar-nasal cartilages with telecanthus, Alar cartilageshypoplasia-coloboma-telecanthus, Pierre Robin syndrome-fetalchondrodysplasia, Dysspondyloenchondromatosis, Achondroplasiaregional-dysplasia abdominal muscle, Osteochondritis Dissecans, FamilialArticular Chondrocalcinosis, Tracheobronchomalacia, Chondritis,Dyschondrosteosis, Maffucci Syndrome, Jequier-Kozlowski-skeletaldysplasia, Chondrodystrophy, Cranio osteoarthropathy, Tietze's syndrome,Hip dysplasia-enchondromata-enchondromata, Bessel-Hagen disease,Chondromatosis (benign), Enchondromatosis (benign), chondrocalcinosisdue to apatite crystal deposition, Meyenburg-Altherr-Uehlinger syndrome,Enchondromatosis-dwarfism-deafness, Astley-Kendall syndrome, Synovialosteochondromatosis, Chondrocalcinosis familial articular, Severeachondroplasia with developmental delay and acanthosis nigricans,Chondrocalcinosis, Keutel syndrome, Stanescu syndrome,Fibrochondrogenesis, Hypochondroplasia,

A “composition” or “pharmaceutical composition” are used interchangeablyherein refers to a composition that usually contains an excipient, suchas a pharmaceutically acceptable carrier that is conventional in the artand that is suitable for administration to a HB-X conjugate to a tissueor subject. In addition, compositions for topical (e.g., oral mucosa,respiratory mucosa) and/or oral administration can form solutions,suspensions, tablets, pills, capsules, sustained-release formulations,oral rinses, or powders, as known in the art and described herein. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, University of theSciences in Philadelphia (2005) Remington: The Science and Practice ofPharmacy with Facts and Comparisons, 21st Ed.

As used herein, the terms “treat,” “treating,” and “treatment” refer tothe alleviation or measurable lessening of one or more symptoms ormeasurable markers of a disease or disorder; while not intending to belimited to such, disease or disorders of particular interest includeautoimmune diseases and myositis. Measurable lessening includes anystatistically significant decline in a measurable marker or symptom. Insome embodiments, treatment is prophylactic treatment.

The term “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result, e.g., a diminishment or prevention ofeffects associated with various disease states or conditions, such asreduce a symptom of an autoimmune disease in the subject. The term“therapeutically effective amount” refers to an amount of an HB-Xconjugate as disclosed herein effective to treat or prevent a disease ordisorder in a mammal, preferably a human. A therapeutically effectiveamount of a HB-X conjugate can vary according to factors such as thedisease state, age, sex, and weight of the subject, and the ability ofthe therapeutic compound X to elicit a desired response in the subject.A therapeutically effective amount is also one in which any toxic ordetrimental effects of the therapeutic agent are outweighed by thetherapeutically beneficial effects. In some embodiments, atherapeutically effective amount is an ‘effective amount”, which is usedherein refers to the amount of therapeutic agent of pharmaceuticalcomposition to alleviate at least one or some of the symptoms of thedisease or disorder. An “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art and is theamount to achieve improvement including, but not limited to, improvedsurvival rate or more rapid recovery, or improvement or elimination ofat least one symptom and other indicator of the disease being treatedwhich are appropriate measures by those skilled in the art. It should benoted that HB-X fusion proteins as disclosed herein can be administeredas a pharmaceutically acceptable salt and can be administered alone oras an active ingredient in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles.

The term “prophylactically effective amount” refers to an amount of aHB-X conjugate which is effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result.

Typically, since a prophylactic dose of HB-X conjugate is administeredto a subject prior to, or at an earlier stage of a disease, and in someembodiments, a prophylactically effective amount is less than thetherapeutically effective amount. A prophylactically effective amount ofa HB-X conjugate is also one in which any toxic or detrimental effectsof the compound are outweighed by the beneficial effects.

As used herein, the terms “prevent,” “preventing” and “prevention” referto the avoidance or delay in manifestation of one or more symptoms ormeasurable markers of a disease or disorder, e.g., of an autoimmunedisease. A delay in the manifestation of a symptom or marker is a delayrelative to the time at which such symptom or marker manifests in acontrol or untreated subject with a similar likelihood or susceptibilityof developing the disease or disorder. The terms “prevent,” “preventing”and “prevention” include not only the avoidance or prevention of asymptom or marker of the disease, but also a reduced severity or degreeof any one of the symptoms or markers of the disease, relative to thosesymptoms or markers in a control or non-treated individual with asimilar likelihood or susceptibility of developing the disease ordisorder, or relative to symptoms or markers likely to arise based onhistorical or statistical measures of populations affected by thedisease or disorder. By “reduced severity” is meant at least a 10%reduction in the severity or degree of a symptom or measurable diseasemarker, relative to a control or reference, e.g., at least 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or even 100% (i.e., nosymptoms or measurable markers).

As used herein, the terms “administering,” and “introducing” are usedinterchangeably herein and refer to the placement of HB-X conjugate ofthe present invention into a subject by a method or route which resultsin at least partial localization of the HB-X conjugate at a desiredsite. The compounds of the present invention can be administered by anyappropriate route which results in an effective treatment in thesubject.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration of HB-X such that it enters the animal's system and,thus, is subject to metabolism and other like processes, for example,subcutaneous administration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation.

The term “reduced” or “reduce” or “decrease” as used herein generallymeans a decrease by a statistically significant amount relative to areference. For avoidance of doubt, “reduced” means statisticallysignificant decrease of at least 10% as compared to a reference level,for example a decrease by at least 20%, at least 30%, at least 40%, atleast t 50%, or least 60%, or least 70%, or least 80%, at least 90% ormore, up to and including a 100% decrease (i.e., absent level ascompared to a reference sample), or any decrease between 10-100% ascompared to a reference level, as that term is defined herein.

The terms “increased” or “increase” as used herein generally mean anincrease by a statically significant amount; such as a statisticallysignificant increase of at least 10%> as compared to a reference level,including an increase of at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100% or more, inclusive, including, for example at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 10-foldincrease or greater as compared to a reference level, as that term isdefined herein.

The term “high” as used herein generally means a higher by a staticallysignificant amount relative to a reference; such as a statisticallysignificant value at least 10% higher than a reference level, forexample at least 20% higher, at least 30% higher, at least 40% higher,at least 50% higher, at least 60% higher, at least 70% higher, at least80% higher, at least 90% higher, at least 100% higher, inclusive, suchas at least 2-fold higher, at least 3-fold higher, at least 4-foldhigher, at least 5-fold higher, at least 10-fold higher or more, ascompared to a reference level.

The term “subject” as used herein refers to any animal in which it isuseful to modulate a response in a tissue targeted by the HB moiety ofthe composition. The subject can be a wild, domestic, commercial orcompanion animal such as a bird or mammal. The subject can be a human.Although in one embodiment of the invention it is contemplated that thetherapeutic compositions as disclosed herein, can also be suitable forthe therapeutic treatment in humans, it is also applicable towarm-blooded vertebrates, e.g., mammals, such as non-human primates,(particularly higher primates), sheep, dog, rodent (e.g., mouse or rat),guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such aschickens, ducks, or turkeys. In some embodiments, the subject is anexperimental animal or animal substitute as a disease model.

The term “pharmaceutically acceptable” refers to compounds andcompositions which may be administered to mammals without unduetoxicity. The term “pharmaceutically acceptable carriers” excludestissue culture medium. Exemplary pharmaceutically acceptable saltsinclude but are not limited to mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like, andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Pharmaceutically acceptable carriers arewell-known in the art. Some pharmaceutically acceptable carriers may beused to provide for sustained release of the compositions describedherein. For example, hyaluronic acid and hyaluronic acid gel forms areused in intra-articular injections, and can be used to provide forsustained release of the HB-X compositions.

As used herein, the terms “treating,” “treatment”, and “to treat” areused to indicate the production of beneficial or desired results, suchas to alleviate symptoms, or eliminate the causation of a disease ordisorder either on a temporary or a permanent basis, slow the appearanceof symptoms and/or progression of the disorder, or prevent progressionof disease. The terms “treat” or “treatment” refer to both therapeutictreatment and prophylactic or preventative measures. Beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of autism, stabilized (i.e., notworsening) state of pathology involvement, delay or slowing of diseaseprogression, amelioration or palliation of the disease state. An“effective regimen” is administered over an effective course (asufficient treatment or amount over a sufficient period of time) toachieve level of desired results. Monitoring efficacy can be done bymethods known in the art for the particular disease or its symptoms.

HB-X Conjugates

An aspect of the present invention as disclosed herein comprises acontrolled release therapeutic composition comprising, for example,(HB-linker)n-Xm-(linker-HB)o, where HB is a heparin binding protein, Xis an active agents such as a therapeutic protein or a portion thereof,or a therapeutic small molecule, and where n, m, and o are integers, andm is at least one and n+o is at least one. In certain embodiments thepharmacokinetics of X may be altered through changes in n or o.

In some embodiments, a HB-X conjugate is HB-Xn, or (HB-linker)n-Xn, andwhere n is an integer of at least 1. The HB can be attached to the N- orC-terminus, or both the N- and C-terminus of the active agent X. In someembodiments, a HB-X conjugate is a recombinant fusion protein comprisinga recombinant HB and the therapeutic protein. In some embodiments, aHB-X conjugate is comprises a HB peptide as disclosed herein and a smallmolecule. In both instances, the HB peptide can be attached (e.g.,conjugated) with an active agent with or without a linker entity. Insome embodiments, the components of a HB-X conjugate can be placed inorder, relative to the N-terminus of the HB portion of the composition:HB-X, X-HB, HB-linker-X, (HB-linker)n-X, X-(linker-HB)n, X-HBn-X,HBn-X-HBn, (HB-linker)n-Xm-(linker-HB)n, HBn-Xm-HBn-Xm, etc.

In some embodiments, a HB-X conjugate can comprise at least 1, or atleast 2, or at least 3, or at least about 4, or at least about 6, or atleast about 7, or at least about 8, or at least about 9, or at leastabout 10, or more than 10 HB peptides of SEQ ID NO: 1-3, 20-22, 41, or82. A HB-X conjugate with more than one HB peptide can have all the sametype of HB peptide (e.g., all HB peptides comprise SEQ ID NO: 2) or cancomprise any combination of different HB peptides from SEQ ID N: 1-3, or20-22. In some embodiments, a HB-X conjugate can comprise at least onelinker associated with a HB peptide, e.g., each HB present in a HB-Xconjugate can be associated with a linker. Alternatively, in someembodiments, where a HB-X conjugate comprises more than one HB peptide,not all HB peptides in a HB-X conjugate need be associated with alinker. The HB peptides can be randomly or non-randomly interspersedbetween a sequence of active agents in a HB-X conjugate, e.g., as anexemplary example, HB-X-X-X-HB-X-X-X-HB, or X-X-HB-X-HB-X-HB-X-X-X-HB-etc. Such a random or non-random interspersion of HB peptides between Xactive entities can occur with or without linkers, as disclosed herein.

Additionally, the composition can comprise a mixture of HB-X constructs,wherein X represents different proteins or small molecules (i.e., acomposition comprising HB-X1 and HB-X2, etc.).

In some embodiments, a HB-X conjugate can comprise at least 1, or atleast 2, or at least 3, or at least about 4, or at least about 6, or atleast about 7, or at least about 8, or at least about 9, or at leastabout 10, or more than 10 active agents (X). Active agents can be atherapeutic protein or peptide as disclosed herein, or a small molecule.

Heparin Binding Proteins (HB)

The present invention relates to proteinaceous heparin-binding motifs(HB) that are linked to or fused to a therapeutic moiety or activeagent, such as a small molecule, cytokine, growth factor, allergenicentity, immunogenic entity or functional portion thereof. In someembodiments, the HB peptide can be selected from peptides having theamino acid residue sequence: KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1);KRKKKGKGLGKKRDPRLRKYK (SEQ ID NO: 2) (C16R); KRKKKGKGLGKKRDPKLRKYK (SEQID NO: 3) (C16K); or KRKKKGKGLGKKRDPSLRKYK (SEQ ID NO: 82) (C16S).

In some embodiments, the HB peptide can be selected from peptides havingthe amino acid residue sequence: MKRKKKGKGLGKKRDPCLRKYK (SEQ ID NO:20);MKRKKKGKGLGKKRDPRLRKYK (SEQ ID NO:21) (C17R); or MKRKKKGKGLGKKRDPKLRKYK(SEQ ID NO:22) (C17K); or MKRKKKGKGLGKKRDPSLRKYK (SEQ ID NO: 41) (C17S).

In some embodiments, a HB portion of the composition is positivelycharged through many lysine and arginine residues; and binds to cellularor tissue proteoglycans which are negatively charged by sulfate groups,allowing for controlled systemic release from the cells or tissues. Inparticular embodiments, a HB is mutated to enhance the positive charge,by replacing the native cysteine residue found at position 16 of the HBhaving the residues of SEQ ID NO: 1 with an arginine (SEQ ID NO: 2)(C16R) or lysine (SEQ ID NO: 3) (C16K). The HB can be repeated with orwithout the inclusion of a peptide linker. Example tandem HB peptideconstructs with linkers be represented HB-linker-HB-X, whereinHB-linker-HB comprises the HB variant of SEQ ID NO: 2 and has the aminoacids: KRKKKGKGLGKKRDPRLRKYKGGGKRKKKGKGLGKKRDPRLRKYK (SEQ ID NO: 4); orHB-linker-HB-linker-HB-X, wherein HB-linker-HB-linker-HB has theresidues: KRKKKGKGLGKKRDPRLRKYKGGGKRKKKGKGLGKKRDPRLRKYKGGGKRKKKGKGLGKKRDPRLRKYK (SEQ ID NO: 5). In some embodiments, the tandem HBpeptide constructs with linkers be represented HB-linker-HB-X, wherein aHB-linker-HB construct that comprises the HB variant of SEQ ID NO: 3 hasthe amino acids: KRKKKGKGLGKKRDPKLRKYKGGGKRKKKGKGLGKKRDPKLRKYK (SEQ IDNO: 23); or HB-linker-HB-linker-HB-X, wherein HB-linker-HB-linker-HBthat comprises the HB variant of SEQ ID NO: 3 has the residues:KRKKKGKGLGKKRDPKLRKYKGGGKRKKKGKGLGKKRDPKLRKYKGGGKRKKKGK GLGKKRDPKLRKYK(SEQ ID NO: 24). In some embodiments, the tandem HB constructs comprisethe same HB construct. In alternative embodiments, it is envisioned thatthe tandem HB peptide constructs can comprise different combinations ofHB peptides, e.g., any combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 82, in any order. As an exemplary example, a tandem HBpeptide construct can comprise SEQ ID NO: 2-linker-SEQ ID NO:3-linker-SEQ ID NO: 3-X. In some embodiments a tandem HB peptideconstruct comprises a combination of HB peptides of SEQ ID NO: 2 and SEQID NO: 3 only.

Linkers

The attachment of a HB peptide to X can be by means of linkers, such as,but not limited to chemical modification, peptide linkers, chemicallinkers, covalent or non-covalent bonds, or protein fusion or by anymeans known to one skilled in the art. The joining can be permanent orreversible. In some embodiments, several linkers can be included inorder to take advantage of desired properties of each linker and eachprotein in the conjugate. Flexible linkers and linkers that increase thesolubility of the conjugates are contemplated for use alone or withother linkers as disclosed herein. Peptide linkers can be linked byexpressing DNA encoding the linker to one or more proteins in theconjugate. Linkers can be acid cleavable, photocleavable and heatsensitive linkers. Methods for conjugation are well known by personsskilled in the art and are encompassed for use in the present invention.

In some embodiments, a HB peptide can be joined to an active agent X,where the active agent is a therapeutic peptide or protein by a peptidelinker. Peptide linkers can be linked by expressing DNA encoding thelinker to one or more proteins in the conjugate. In some embodiments, apeptide linker is GGG. Optionally the linker peptide will be joined atone or both of the amino terminus and carboxy terminus of the HB peptidewith a short flexible linker, e.g. comprising at least about 2, 3, 4 ormore glycine, serine and/or alanine residues. In some embodiments, onesuch linker comprises the motif (GGGGS) (SEQ ID NO: 42), and may bepresent in one or more copies. In some embodiments, the linker comprisespositively charged amino acid residues.

According to the present invention, the HB peptide can be linked to anactive agent via any suitable means, as known in the art, see forexample U.S. Pat. Nos. 4,625,014, 5,057,301 and 5,514,363, which areincorporated herein in their entirety by reference.

A large variety of methods for conjugation of a HB peptide as disclosedherein with X, e.g., a first fusion partner (e.g. a therapeutic proteinor peptide) are known in the art. Such methods are e.g. described byHermanson (1996, Bioconjugate Techniques, Academic Press), in U.S. Pat.Nos. 6,180,084 and 6,264,914 which are incorporated herein in theirentirety by reference and include e.g. methods used to link haptens tocarriers proteins as routinely used in applied immunology (see Harlowand Lane, 1988, “Antibodies: A laboratory manual”, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). It is recognized that, insome cases, a HB peptide and/or therapeutic protein or peptide can loseefficacy or functionality upon conjugation depending, e.g., on theconjugation procedure or the chemical group utilized therein. However,given the large variety of methods for conjugation the skilled person isable to find a conjugation method that does not or least affects theefficacy or functionality of the entities, such as an HB peptide and thetherapeutic peptide which is to be conjugated.

In some embodiments a HB peptide can be conjugated to an active agent(X) by cross-linking Crosslinking reagents include glutaraldehyde (GAD),bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE),N-hydroxysuccinimide (NHS), and a water soluble carbodiimide, preferably1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). As is known to theskilled artisan, any crosslinking chemistry can be used, including, butnot limited to, thioether, thioester, malimide and thiol,amine-carboxyl, amine-amine, and others listed in organic chemistrymanuals, such as, Elements of Organic Chemistry, Isaak and HenryZimmerman Macmillan Publishing Co., Inc. 866 Third Avenue, New York,N.Y. 10022.

Other linkage approaches to conjugate the HB peptide to the activeagent, include but are not limited to aminocaproic horse radishperoxidase (HRP) or a heterobiofunctional cross-linker, e.g., carbonylreactive and sulfhydryl-reactive cross-linker Heterobiofunctional crosslinking reagents usually contain two reactive groups that can be coupledto two different function targets on proteins and other macromoleculesin a two or three-step process, which can limit the degree ofpolymerization often associated with using homo-biofunctionalcross-linkers. Such multistep protocols can offer a great control ofconjugate size and the molar ratio of components.

In some embodiments, a HB peptide is conjugated to a nucleic acid activeagent, e.g., RNAi agent or miRNA agent using a protamine linker, asdisclosed in the U.S. Patent Application Publication Nos. US2002/0132990and US2004/0023902, which are incorporated herein in their entirety byreference. In particular, where a linker is a protamine or protaminelike agent, the methods, regents and reference that describe thepreparation of protamine associated with a HB peptide are disclosed inU.S. Provisional Application 60/957,023 and US Patent ApplicationsUS2007/012152, and US 2010/0209440, which are each incorporated hereinin their entirety by reference. In some embodiments, a protamine linkerencompassed for use in the present invention comprises SEQ ID NO: 1-6disclosed in US 2010/0209440.

Suitable methods for conjugation of a HB peptide as disclosed hereinwith X (e.g., a first fusion partner (e.g. a therapeutic protein orpeptide) include e.g. carbodimide conjugation (Bauminger and Wilchek,1980, Meth. Enzymol. 70: 151-159). Alternatively, a moiety can becoupled to a targeting agent as described by Nagy et al., Proc. Natl.Acad. Sci. USA 93:7269-7273 (1996), and Nagy et al., Proc. Natl. Acad.Sci. USA 95: 1794-1799 (1998), each of which are incorporated herein byreference. Another method for conjugating one can use is, for examplesodium periodate oxidation followed by reductive alkylation ofappropriate reactants and glutaraldehyde crosslinking.

One can use a variety of different linkers to conjugate a HB peptide asdisclosed herein with X (e.g., a first fusion partner (e.g. atherapeutic protein or peptide), for example but not limited toaminocaproic horse radish peroxidase (HRP) or a heterobiofunctionalcross-linker, e.g. carbonyl reactive and sulfhydryl-reactivecross-linker. Heterobiofunctional cross linking reagents usually containtwo reactive groups that can be coupled to two different functiontargets on proteins and other macromolecules in a two or three-stepprocess, which can limit the degree of polymerization often associatedwith using homobiofunctional cross-linkers. Such multi-step protocolscan offer a great control of conjugate size and the molar ratio ofcomponents.

In some embodiments, a linker is a immunoglobulin hinge region linker asdisclosed in U.S. Pat. Nos. 6,165,476, 5,856,456, US Application2010/0063258 and International Application WO2012/142515, each of whichare incorporated herein in their entirety by reference.

In some embodiments, a HB-X fusion protein can be produced in acell-free system as disclosed in U.S. Application 2010/0063258, which isincorporated herein in its entirety by reference. Exemplary linkersequences include for example: (i) the tail region of the membrane longisoform of IgA 1 (malL): SCSVADWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO:43), (ii) the tail region of the membrane variant long isoform of IgA 1(ma 1 L with extra cys):

SCCVADWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 44), (iii) the tail regionof the membrane short isoform of IgA 1 (mals with 6 amino acidN-terminal deletion):

DWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 45), (iv) the tail region of themembrane bound form of IgA2: SCCVADWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO:46), (v) the tail region of the membrane bound form of IgD:YLAMTPLIPQSKDENSDDYTTFDDVGS (SEQ ID NO: 47), (vi) the tail region of themembrane-bound form of IgE: ELDVCVEEAEGEAPW (SEQ ID NO: 48), (vii) thetail region of the membrane bound form of IgG: ELQLEESCAEAQDGELDG (SEQID NO: 49), and (viii) the tail region of the membrane bound form of IgMEGEVSADEEGFEN (SEQ ID NO: 50).

In other embodiments, a linker sequence is derived from the tail segmentof a secretory or soluble form of an immunoglobulin. Exemplary linkersequences include for example: (i) the tail region of the soluble formof IgAl: KPTHVNVSVVMAEVDGTCY (SEQ ID NO: 51), (ii) the tail region ofthe soluble form of IgA2: KPTHVNVSVVMAEVDGTCY (SEQ ID NO: 52), (iii) thetail region of the soluble form of IgD: YVTDHGPMK (SEQ ID NO: 53), and(iv): the tail region of the soluble form of IgM: PTLYNVSLVMSDTAGTCY(SEQ ID NO: 54).

In certain embodiments, it may be desirable to have a linker sequencecontaining a free cysteine residue in order to permit the formation of adisulfide bond between linkers thereby forming dimers of the HB fusionproteins. In other embodiments, it may be desirable to alter the linkersequences to remove free cysteine residues, e.g., by mutating one ormore cysteine residues in a linker to another residue, such as a serine,alanine or glycine. Examples of linker sequences derived from the tailregions of membrane bound immunoglobulins that have been altered toremove free cysteine residues include: (i)SXSVADWQMPPPYVVLDLPQETLEEETPGAN, wherein X is serine, alanine or glycine(SEQ ID NO: 55), (ii) SXXVADWQMPPPYVVLDLPQETLEEETPGAN, wherein each X isindependently selected from serine, alanine or glycine (SEQ ID NO: 56),(iii) SXXVADWQMPPPYVVLDLPQETLEEETPGAN, wherein each X is independentlyselected from serine, alanine or glycine (SEQ ID NO: 57), (iv)ELDVXVEEAEGEAPW, wherein X is serine, alanine or glycine (SEQ ID NO:58), and (v) ELQLEESXAEAQDGELDG, wherein X is serine, alanine or glycine(SEQ ID NO: 59). Examples of linker sequences derived from the tailregions of secretory forms of immunoglobulins that have been altered toremove free cysteine residues include: (i) KPTHVNVSVVMAEVDGTXY, whereinX is serine, alanine or glycine (SEQ ID NO: 60), (ii)KPTHVNVSVVMAEVDGTXY, wherein X is serine, alanine or glycine (SEQ ID NO:61), and (iii) PTLYNVSLVMSDTAGTXY, wherein X is serine, alanine orglycine (SEQ ID NO: 62).

Active Agents—Therapeutic Proteins to Conjugate to a HB Peptide

The importance and usefulness of the compositions described herein areexemplified in the application of the composition in cartilage repair,wherein the fusion protein is HB-IGF-1. Traumatic injuries to the joint,such as those involving anterior cruciate ligament (ACL) rupture lead toan increased risk for development of osteoarthritis. Furthermore, thisrisk may not be resolved by surgical restoration of function (Lohmanderet al., 35 Am. J. Sports Med. 1756 (2007)), which may be related to theinitial inflammatory and catabolic response following joint injury.Lohmander et al., 42 Arthritis Rheum. 534 (1999); Lohmander et al., 48Arthritis Rheum. 3130 (2003); Irie et al., 10 Knee 93 (2003).Therapeutic interventions in this time period may be particularlyimportant for opposing these catabolic processes and promoting cartilagerepair.

IGF-1 is the prototypical circulating factor that stimulates cartilagebiosynthesis. Daughaday et al., 19 J. Clin. Endocrinol. Metab. 743(1959); McQuillan et al., 240 Biochem. J. 423 (1986); Jones & Clemmons,16 Endocrine Rev. 3 (1995). It also acts to oppose catabolic stimuli.Luyten et al., 267 Arch. Biochem. Biophys. 416 (1988); Tyler, 260Biochem. J. 543 (1989). As a result, investigators have long sought touse IGF-1 as a therapy for cartilage repair. Trippel, 43 J. Rheumatol.Suppl. 129 (1995). Local delivery of IGF-1 and other growth factors isseverely limited, however, by their short half-life in the joint.Investigators have developed options for gene therapy with IGF-1 and forIGF-1 encapsulated in hydrogels to allow for long-term controlledrelease to the joint cartilage. Although promising, these techniqueshave been slow to reach clinical trials. Evans et al., 7 Nat. Rev.Rheum. 244 (2011).

An example rat IGF-1 protein has the amino acid residues:

GPETLCGAELVDALQFVCGPRGFYFNKPTGYGSSIRRAPQTGIVDECCFRSCDLR RLEMYCAPLKPTKSA(SEQ ID NO: 6). See, e.g., Tokunou et al., 22 FASEB J. 1886 (2008).

An example human IGF-1 has the amino acid residues:

GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 7). See alsoGene ID: 3489 (human IGF1). Another example human IGF-1 (variant) hasthe amino acid residue sequence:GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM YCAPLKPAKSA.(SEQ ID NO: 8). A variant of human IGF-1 that has biological activity(see WO 92/03477; GenBank: CAA01451.1) has the amino acid residues:megpeticgaelvdalqfvcgdrgfyfnkptgygsssrrapqtgivdeccfrscdtrrlemycaplkpaksa(SEQ ID NO: 9).

It is known that truncation of the N-terminus of IGF-1 retainsbiological activity, e.g., the deletion of N-terminal amino acids GPE.Accordingly, in some embodiments, a human IGF-1 (variant) encompassedfor use in the present invention has the amino acid residue sequence:

TLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLE MYC APLKPAKSA.(SEQ ID NO: 63).

Thus, in some embodiments a recombinant HB-linker-HB-X fusion protein,wherein HB is a C16R variant, the linker is GGG, and X is a variant ofhuman IGF-1 of SEQ ID NO: 7, has the amino acid sequence:

KRKKKGKGLGKKRDPRLRKYKGGGKRKKKGKGLGKKRDPRLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 10); another recombinantHB-linker-HB-linker-HB-X, wherein HB is a C16R variant, the linker isGGG, and X is a variant of human IGF-1 (SEQ ID NO: 7) can be depicted:KRKKKGKGLGKKRDPRLRKYKGGGKRKKKGKGLGKKRDPRLRKYKGGGKRKKKGKGLGKKRDPRLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 11).

In some embodiments, a variant of IGF-1 of SEQ ID NO: 7 can besubstituted for SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, theHB C16R (e.g., SEQ ID NO: 2) can be substituted for a HB C17R (e.g.,MKRKKKGKGLGKKRDPRLRKYK; SEQ ID NO: 21) An example fusion of HB C17R withfull-length human IGF-1 (of SEQ ID NO: 7) has the amino acids:MKRKKKGKGLGKKRDPRLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNA SRGSA (SEQ IDNO: 12).

In some embodiments, an example fusion of HB C16R with full-length humanIGF-1 (of SEQ ID NO: 7) (e.g., HB-IGF) has the amino acids:KRKKKGKGLGKKRDPRLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASR GSA (SEQ IDNO: 16).

In some embodiments, HB C16R (e.g., SEQ ID NO: 2) or HB C17R (SEQ ID NO:21) can be substituted for HB C16K (SEQ ID NO: 3) or HB C17K(MKRKKKGKGLGKKRDPKLRKYK; SEQ ID NO: 22). For example, in anotherexample, a fusion of HB C17K with full-length human IGF-1 (e.g., SEQ IDNO: 7) has the amino acids:MKRKKKGKGLGKKRDPKLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNA SRGSA (SEQ IDNO: 13). Truncations of human IGF-1 corresponding to SEQ ID NO: 8 fusedto HB C17R has the amino acid sequence of:MKRKKKGKGLGKKRDPRLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO:14) orKRKKKGKGLGKKRDPRLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO:18), and a fusion of HBC17K with mature human IGF-1:MKRKKKGKGLGKKRDPKLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO:15)

Variants of the foregoing HB-fusion proteins can be constructed thatlack the initial N-terminal methionine on the HB peptide, e.g.,comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, i.e.,KRKKKGKGLGKKRDPRLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASR GSA (SEQ IDNO: 16); KRKKKGKGLGKKRDPKLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASR GSA (SEQ IDNO: 17); KRKKKGKGLGKKRDPRLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO:18); andKRKKKGKGLGKKRDPKLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO:19).

Table 1 discloses examples of different embodiments of fusion of HBpeptide to an agent. As an exemplary agent (e.g., X), IGF-1 is used:

IGF- HB 1variant peptide Linker (e.g., X) Sequence HB- C16R GGG SEQ IDKRKKKGKGLGKKRDPRLRKYK GGG KRKKKGKGLGKKRDP linker- (SEQ ID NO: 7RLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRR HB-X NO: 2)APQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 10); HB- C16R GGG SEQ IDKRKKKGKGLGKKRDPRLRKYK GGG KRKKKGKGLGKKRDP linker- (SEQ ID NO: 7 RLRKYKGGG KRKKKGKGLGKKRDPRLRKYKGPETLCGAELV HB- NO: 2)DALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLR linker-RLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA HB-X (SEQ ID NO: 11) HB-XC17R — SEQ ID MKRKKKGKGLGKKRDPRLRKYKGPETLCGAELVDALQFVCG (SEQ ID NO: 7DRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPL NO: 21)KPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 12). HB-X C17K — SEQ IDMKRKKKGKGLGKKRDP K LRKYKGPETLCGAELVDALQFVCG (SEQ ID NO: 7DRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPL NO: 22)KPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 13) HB-X C17R — SEQ IDMKRKKKGKGLGKKRDP R LRKYKGPETLCGAELVDALQFVCG (SEQ ID NO: 8DRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPL NO: 21)KPAKSA (SEQ ID NO: 14) HB-X C17K — SEQ IDMKRKKKGKGLGKKRDPKLRKYKGPETLCGAELVDALQFVCG (SEQ ID NO: 8DRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPL NO: 22)KPAKSA (SEQ ID NO: 15) HB-X C16R — SEQ ID KRKKKGKGLGKKRDP RLRKYKGPETLCGAELVDALQFVCGD (SEQ ID NO: 7RGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLK NO: 2)PAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 16); HB-X C16K — SEQ IDKRKKKGKGLGKKRDP K LRKYKGPETLCGAELVDALQFVCGD (SEQ ID NO: 7RGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLK NO: 3)PAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 17); HB-X C16R — SEQ IDKRKKKGKGLGKKRDP R LRKYKGPETLCGAELVDALQFVCGD (SEQ ID NO: 8RGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLK NO: 2)PAKSA (SEQ ID NO: 18); HB-X C16K — SEQ IDKRKKKGKGLGKKRDPKLRKYKGPETLCGAELVDALQFVCGD (SEQ ID NO: 8RGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLK NO: 3)PAKSA (SEQ ID NO: 19). HB- C16R GGG SEQ ID KRKKKGKGLGKKRDPRLRKYK GGGKRKKKGKGLGKKRDP linker- (SEQ ID NO: 8RLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRR HB-X NO: 2)APQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO: 25); HB- C16R GGG SEQ IDKRKKKGKGLGKKRDPRLRKYK GGG KRKKKGKGLGKKRDP linker- (SEQ ID NO: 8 RLRKYKGGG KRKKKGKGLGKKRDPRLRKYKGPETLCGAELV HB- NO: 2)DALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLR linker-RLEMYCAPLKPAKSA (SEQ ID NO: 26) HB-X HB- C16K GGG SEQ IDKRKKKGKGLGKKRDPKLRKYK GGG KRKKKGKGLGKKRDP linker- (SEQ ID NO: 7KLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRR HB-X NO: 3)APQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 27); HB- C16K GGG SEQ IDKRKKKGKGLGKKRDPKLRKYK GGG KRKKKGKGLGKKRDP linker- (SEQ ID NO: 7 KLRKYKGGG KRKKKGKGLGKKRDPKLRKYKGPETLCGAEL HB- NO: 3)VDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDL linker-RRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGS HB-X A (SEQ ID NO: 28) HB-C16K GGG SEQ ID KRKKKGKGLGKKRDPKLRKYK GGG KRKKKGKGLGKKRDP linker-(SEQ ID NO: 8 KLRKYKGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRR HB-X NO: 3)APQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO: 29); HB- C16K GGG SEQ IDKRKKKGKGLGKKRDPKLRKYK GGG KRKKKGKGLGKKRDP linker- (SEQ ID NO: 8 KLRKYKGGG KRKKKGKGLGKKRDPKLRKYKGPETLCGAEL HB- NO: 3)VDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDL linker-RRLEMYCAPLKPAKSA (SEQ ID NO: 30). HB-X HB-X C16R — SEQ IDKRKKKGKGLGKKRDP R LRKYKTLCGAELVDALQFVCGDRGF (SEQ ID NO: 63YFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPA NO: 2)KSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 64); HB-X C16K — SEQ IDKRKKKGKGLGKKRDP K LRKYKTLCGAELVDALQFVCGDRGF (SEQ ID NO: 63YFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPA NO: 3)KSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 65); HB-X C16R — SEQ IDKRKKKGKGLGKKRDP R LRKYKTLCGAELVDALQFVCGDRGF (SEQ ID NO: 63YFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPA NO: 2) KSA (SEQ ID NO: 66);HB-X C16K — SEQ ID KRKKKGKGLGKKRDPKLRKYKTLCGAELVDALQFVCGDRGF (SEQ IDNO: 63 YFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPA NO: 3)KSA (SEQ ID NO: 67). HB-X C16R GGG SEQ ID KRKKKGKGLGKKRDP RLRKYKGGGTLCGAELVDALQFVCGD (SEQ ID NO: 63RGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLK NO: 2)PAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 68); HB-X C16K GGG SEQ IDKRKKKGKGLGKKRDP K LRKYKGGGTLCGAELVDALQFVCGD (SEQ ID NO: 63RGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLK NO: 3)PAKSARSVRAQRHTDMPKTQKEVHLKNASRGSA (SEQ ID NO: 69); HB-X C16R GGG SEQ IDKRKKKGKGLGKKRDP R LRKYKGGGTLCGAELVDALQFVCGD (SEQ ID NO: 63RGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLK NO: 2)PAKSA (SEQ ID NO: 70); HB-X C16K GGG SEQ IDKRKKKGKGLGKKRDPKLRKYKGGGTLCGAELVDALQFVCGD (SEQ ID NO: 63RGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLK NO: 3)PAKSA (SEQ ID NO: 71).

It is also encompassed that the HB peptide can be located at theN-terminus or C-terminus or at the N- and C-terminus of the activeagent, e.g., an active agent for example, such as IGF-1. In someembodiments, the HB peptide can be located at the N-terminus orC-terminus or at the N- and C-terminus of the active agent with orwithout a linker at each transition between the HB peptide and theactive agent. Accordingly, variations of the sequences in Table 1 (e.g.,SEQ ID NO: 12-71) are encompassed where the HB is located at theC-terminus instead of the N-terminus, and where there is the presence orabsence of a linker between the IGF-1 protein and the sequence of theC-terminus HB. In additional embodiments, variations of the sequences inTable 1 (e.g., SEQ ID NO: 12-71) are encompassed where the HB is locatedat both the C- and N-terminus, and where there is the presence orabsence of a linker between the IGF-1 protein and the sequence of the C-or N-terminus HB. Any combination of a HB peptide selected from thegroup of SEQ ID NO: 1-3 or 30-22 can be used in any combination of anactive agent, with or without the presence of a linker protein, wherethe HB peptide can be located at the N- and/or C-terminus of the activeagent, and there can be one or multiple HB peptide-linkers attached tothe N- and/or C-terminus of the active agent. For example, in someembodiments, the fusion or conjugate can comprise(HB-linker)n-Xm-(linker-HB)0, where n, m, and o are integers, and m isat least one and n+o is at least one.

In certain aspects, compounds and methods of the invention may relate tomodifying the pharmacokinetic properties, including the retention intissue, the release to systemic circulation, and the systemic half-lifeof a therapeutic moiety by incorporating one or more of the HB domainsdescribed above. For example, the inventors have shown that a C17Smodified HB-IGF-1 fusion protein remains detectable in skin tissue forat least 24 hours after intradermal injection where IGF-1 alone wasundetectable after 6 hours. In another example, the inventors found thatmore than three times the amount of C17S modified HB-IGF-1 fusionprotein was retained in skin tissue than IGF-1 alone 24 hours aftersubcutaneous injection.

In certain aspects, compounds and methods of the invention may relate tomodifying the pharmacokinetic properties, including the retention intissue, the release to systemic circulation, and the systemic half-lifeof a therapeutic moiety by incorporating one or more of the HB domainsdescribed above. For example, the inventors have shown that a C17Smodified HB-IGF-1 fusion protein remains detectable in skin tissue forat least 24 hours after intradermal injection where IGF-1 alone wasundetectable after 6 hours. In another example, the inventors found thatmore than three times the amount of C17S modified HB-IGF-1 fusionprotein was retained in skin tissue than IGF-1 alone 24 hours aftersubcutaneous injection. Accordingly, HB fused therapeutic moieties ofthe invention, such as C17S modified HB-IGF-1 may, be released intosystemic circulation at a slower rate than therapeutic moieties alone,such as IGF-1.

In certain aspects, compounds and methods of the invention may relate toadministration of HB fused therapeutic moieties to the skin of patientsby, for example, intradermal injection or subcutaneous injection.

In certain aspects, compounds may include an immunogenic entity adaptedto elicit an immune response and prevent disease through immunization.Immunogenic entities may include an immunogenic entity adapted to elicitan immune response to diseases including, for example, cancer, anthrax,measles, rubella, cholera, meningococcal disease, influenza, diphtheria,mumps, tetanus, hepatitis A, pertussis, tuberculosis, hepatitis B,pneumococcal disease, typhoid fever, hepatitis E, poliomyelitis,tick-born encephalitis, haemophilus influenzae type b, rabies, varicellaand herpes zoster (shingles), human papilloma-virus, rotavirusgastroenteritis, yellow fever, and Japanese encephalitis.

In some embodiments, the immunogenic entity which is linked to at leastone HB peptide useful in the methods and compositions as disclosedherein is a pathogenic antigen. Examples of such pathogens which expressantigens for use in the invention herein include, but are not limited toMyocobacterium tuberculosis (TB), Herpes simplex virus type-1, Herpessimplex virus type-2, HBV, Cytomegalovirus, Epstein-Barr virus,Varicella-zoster virus, Human herpes virus 6, Human herpes virus 7,Human herpes virus 8, Variola virus, Vesicular stomatitis virus,Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A,Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus,Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus,Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Yellow fevervirus, Ebola virus, Marburg virus, Lassa fever virus, Eastern EquineEncephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitisvirus, Murray Valley fever virus, West Nile virus, Rift Valley fevervirus, Rotavirus A, Rotavirus B. Rotavirus C, Sindbis virus, HumanT-cell Leukemia virus type-1, Hantavirus, Rubella virus, Rinderpest,Rhinovirus, Echovirus, Papova virus, Echinovirus, Arbovirus, HumanImmunodeficiency virus type I or type II and Simian Immunodeficiencyvirus. In some embodiments, the immunogenic entity is an antigenexpressed by the pathogen Myocobacterium tuberculosis, for example, onesuch antigen is a TB-specific antigen, such as a TB1 (CFP10) polypeptideor a fragment thereof or a TB2 (ESAT6) polypeptide or a fragmentthereof. In specific embodiments, the immunogenic entity fused to atleast one HB peptide is a cancer antigen.

In certain aspects, compounds and methods of the invention as describedherein where HB is linked to immunogenic entity may further comprises anadjuvant. Adjuvants are a heterogeneous group of substances that enhancethe immunological response against an antigen that is administeredsimultaneously.

The term “adjuvant” as used herein refers to any agent or entity whichincreases the antigenic response or immune response by a cell to theimmunogenic entity or antigen fused to HB.

In some instances, adjuvants are added to a vaccine to improve theimmune response so that less vaccine is needed. Adjuvants serve to bringthe antigen—the substance that stimulates the specific protective immuneresponse—into contact with the immune system and influence the type ofimmunity produced, as well as the quality of the immune response(magnitude or duration). Adjuvants can also decrease the toxicity ofcertain antigens; and provide solubility to some vaccine components.Almost all adjuvants used today for enhancement of the immune responseagainst antigens are particles or form particles together with theantigen. In the book “Vaccine Design—the subunit and adjuvant approach”(Ed: Powell & Newman, Plenum Press, 1995) almost all known adjuvants aredescribed both regarding their immunological activity and regardingtheir chemical characteristics. The type of adjuvants that do not formparticles are a group of substances that act as immunological signalsubstances and that under normal conditions consist of the substancesthat are formed by the immune system as a consequence of theimmunological activation after administration of particulate adjuvantsystems.

In certain aspects, compounds and compositions as described herein thatare for use as a vaccine or to simulate the immune system furthercomprise an adjuvant. Examples of adjuvants are well known in the artand include, but are not limited to, mineral salts, e.g., aluminumhydroxide and aluminum or calcium phosphate gels; surface activesubstances such as lysolecithin, pluronic polyols, polyanions; otherpeptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum. Commonly used adjuvants include, forexample, but are not limited to, monoglycerides and fatty acids (e.g. amixture of mono-olein, oleic acid, and soybean oil); oil emulsions andsurfactant based formulations, e.g., MF59 (microfluidised detergentstabilised oil-in-water emulsion), QS21 (purified saponin), Detox-PC,MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B,Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN,Betafectin, Alum, AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21),Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion);particulate adjuvants, e.g., virosomes (unilamellar liposomal vehiclesincorporating influenza haemagglutinin), AS04 ([SBAS4] Al salt withMPL), ISCOMS (structured complex of saponins and lipids), polylactideco-glycolide (PLG); microbial derivatives (natural and synthetic), e.g.,monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton),AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidalimmunostimulators able to self organize into liposomes), OM-174 (lipid Aderivative), CpG motifs (synthetic oligonucleotides containingimmunostimulatory CpG motifs), modified LT and CT (genetically modifiedbacterial toxins to provide non-toxic adjuvant effects); endogenoushuman immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can beadministered either as protein or plasmid encoded), Immudaptin (C3dtandem array) and inert vehicles, such as gold particles. In someembodiments, suitable adjuvants include, but are not limited to, LTR72(a mutant of E. coli heat-labile enterotoxin with partial knockout ofADP-ribosyltransferase activity), polyphosphazine adjuvant, interleukinssuch as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12, interferons suchas alpha-interferon and gamma-interferon, tumor necrosis factor (TNF),platelet derived growth factor (PDGF), GCSF, granulocyte-macrophagecolony-stimulating factor (GM-CSF), epidermal growth factor (EGF), andthe like. Examples of adjuvants capable of stimulating cellular immuneresponses include cytokines secreted by helper T cells called Th1 cells,e.g., interleukin-2 (IL-2), interleukin-4, interleukin-12 (IL-12) andinterleukin-18, fusion proteins having one of such Th1 type cytokines(e.g., IL-2) fused to the Fc portion of immunoglobulin G (IgG),interferons such as alpha-interferon, beta-interferon andgamma-interferon, and chemokines that attract T cells to infectedtissues. Non-coding, ISS-enriched plasmid DNAs or ISS oligonucleotides(ISS-ODNs) can also be used in the present invention as adjuvants toenhance cellular immunity. Newer adjuvants are described in U.S. Pat.No. 6,890,540, U.S. Patent Application No. 2005/0244420, andPCT/SE97/01003, the contents of which are incorporated herein byreference in their entirety.

Using particulate systems as adjuvants, the antigens are associated ormixed with or into a matrix, which has the characteristics of beingslowly biodegradable. Care must be taken to ensure that that thematrices do not form toxic metabolites. Preferably, the main kinds ofmatrices used are mainly substances originating from a body. Theseinclude lactic acid polymers, poly-amino acids (proteins),carbohydrates, lipids and biocompatible polymers with low toxicity.Combinations of these groups of substances originating from a body orcombinations of substances originating from a body and biocompatiblepolymers can also be used. Lipids are the preferred substances sincethey display structures that make them biodegradable as well as the factthat they are a critical element in all biological membranes.

In various embodiments, compounds may include an allergenic entity. Anallergenic entity is a type of antigen that produces in an abnormalimmune response in the subject to a harmless entity or commonenvironmental antigen. Without wishing to be bound by theory, anallergenic entity typically stimulates an inappropriate immune responsenormally induced in response to pathogenic antigens. More specifically,an allergenic entity can induce an inappropriate immunoglobulin (e.g.,IgE) response to a normally harmful environmental antigen leading totype-1 hypersensitivity reaction. Allergenic entities linked to aheparin binding peptide may be administered to a patient via, forexample, intradermal injection or subcutaneous injection. Controlledrelease of the allergen into systemic circulation, increased retentionin the patient's skin, and prolonged exposure to Langerhans cells in thepatient's dermis may induce tolerance of the allergen with less frequentinjections. Without wishing to be bound by theory, one or moreallergenic entity can be linked to a heparin binding peptide as asuppression immunotherapy to induce immune tolerance, which is a processby which the body naturally does not launch an immune system attack onits own tissues. Immune tolerance therapies seeks to modulate the immuneresponse so that the body stops mistakenly attacking its own organs orcells in autoimmune disease or accepts foreign tissue in organtransplantation. A brief treatment with the allergenic entity linked toa HB peptide could reduce or eliminate the subject having an allergicreaction to a specific allergenic entity, or in the case oftransplantation, the need for lifelong immunosuppression and associatedside effects, or preserve the body's own function, at least in part, incases of type 1 diabetes or other autoimmune disorders. Allergenicentities may be adapted to induce patient immune tolerance of allergenssuch as pollen (e.g., grass, weeds and tree pollen), pet dander, dustmites, airborne molds, tree nuts, peanuts, legumes, soybeans, fruits,milk, eggs, albumen, fish, shellfish, sesame, urushiol, penicillin anddrug, latex, wool, mold spores, honey bee venom, mosquito stings, yellowjacket venom, hornet venom, wasp venom, and fire ant venom. Otherallergenic entities are encompassed in the present invention, e.g.,allergens in autoimmune diseases, for example, diabetes mellitus type 1,multiple sclerosis, or acquired food allergens, drug allergens, seasonalallergens and the like.

The inventors have previously demonstrated an approach to stimulatingcartilage biosynthesis uses an engineered rat IGF-1 protein fused with arat heparin-binding domain, where the heparin-binding IGF-1 (HB-IGF-1)fusion protein is retained in cartilage after intra-articular injection.(Miller et al., 62 Arth. Rheum. 3686 (2010)). Herein, instead of usingthe wild-type rat HB peptide as used Miller, the inventors have modifiedthe human wild-type HB peptide (e.g., corresponding herein to SEQ ID NO:1), and surprisingly demonstrate that the HB constructs of SEQ ID NO: 2or 3 as disclosed herein (where the C16 of SEQ ID NO: 1 or C17 of SEQ IDNO: 20 is changed from a cysteine (C) to an arginine (R) or lysine (K))results in both (i) a significantly increased expression and yield ofthe HB-IGF-1 fusion protein and (ii) increased retention of theHB-fusion protein in the tissue of interest. Accordingly, the novelmutations in the wild-type human HB peptide to change C16 (of SEQ IDNO: 1) or C17 (of SEQ ID NO: 20) to enhance the positive chargesurprisingly resulted in an unexpected increase in the expression andproduction of HB-X fusion proteins.

The present specification thus demonstrated the kinetics of a HB-IGF-1fusion protein comprising SEQ ID NO: 2 or SEQ ID NO: 3 afterintra-articular injection, and shows functional stimulation of HB-IGF-1on joint cartilage in vivo, and demonstrates therapeutic efficacy invivo of HB-IGF-1 in a rat model of joint injury-induced arthritis.

Accordingly, in some embodiments, a HB peptide of SEQ ID NO: 1-3, 20-22,41, or 82, particularly a HB-X conjugate comprising at least one or acombination of SEQ ID NO: 2, 3, 21 or 22, conjugated to an active agent,such as but not limited to an IGF-1 protein or functional fragmentthereof (e.g., any IGF-1 variant selected from the sequences from thegroup of: SEQ ID NO: 6-9 or 63) is encompassed for use in the presentinvention in methods to treat a cartilage-related disease or disorder.

The inventors demonstrate herein that surprisingly, unlike IGF-1 alone(e.g., not fused to an agent), not only is a HB-IGF-1 fusion protein asdisclosed herein retained in extracellular matrix of the cartilage afterintra-articular injection, it is still able to stimulate cells in thecartilage and is therapeutically effective. More specifically, todetermine the kinetics of HB-IGF-1 binding to cartilage in vivo, jointtissues were harvested after injection and tissue extracts analyzed byWestern analysis (FIG. 1). Two days after injection of IGF-1, there wasno detectable IGF-1 remaining in any of the joint tissues harvested. Incontrast, HB-IGF-1 was retained in both articular and meniscalcartilages, but not in patellar tendon. A similar result was observedfour days after injection. By 6 to 8 days after injection, HB-IGF-1 wasstill detectable in the cartilage extracts but the immunoreactive bandswere faint and more variable. These results demonstrate that, unlikeIGF-1, the present HB-IGF-1 fusion protein is retained in articularcartilage for up to 8 days after intra-articular injection. Thepharmacokinetics of IGF-1 in serum are shown in FIG. 2, indicating thatintra-articularly injected HB-IGF-1 had markedly reduced leakage intothe systemic circulation compared with unmodified IGF-1.

Additionally, the HB-IGF-1 produced sustained stimulation of cartilagebiosynthesis in vivo. HB-IGF-1 remains able to activate cellular IGF-1receptors in vivo despite its increased binding to chondroitin sulfatein the cartilage extracellular matrix. Sulfate incorporation intomeniscal cartilage harvested after injection was measured and normalizedto incorporation after injection of saline only (FIG. 3). Two days afterinjection, HB-IGF-1 stimulated a significantly higher rate of sulfateincorporation than did IGF-1 (HB-IGF: 2.10±0.52; IGF: 0.49±0.11;N=4-5/group; P=0.032). Four days after injection, sulfate incorporationremained significantly higher in the HB-IGF-1 group than in the IGF-1group (HB-IGF: 1.82±0.09; IGF: 1.05±0.21; N=5/group; P=0.011).

Further, HB-IGF-1 protects cartilage in vivo after transection of themedial meniscus. More specifically, HB-IGF-1 is efficacious in a ratmodel of surgically induced OA. Rats were subjected to medial meniscaltear (MMT) surgery and injected weekly with HB-IGF, IGF, or saline.Three weeks after MMT surgery, histological assessment of kneeosteoarthritis (OA) was performed. For the primary outcome measure (FIG.4), the overall OARSI score was significantly lower in the joints of theanimals treated with HB-IGF-1 compared to control animals treated withIGF-1 (HB-IGF: 12.9±1.5; IGF: 18.7±1.2; N=9-10/group; P=0.008).Significant differences between HB-IGF-1 and IGF-1 treated knees werealso observed on secondary analyses of total degeneration width andfull-thickness cartilage loss, as shown in Table 2:

TABLE 2 Cartilage analysis in rat MMT model. (Results are shown as mean± SEM.) Saline IGF-1 HB-IGF-1 (n = 8) (n = 9) (n = 10) Surface cartilageloss 174 ± 56 207 ± 39 120 ± 41 Full-thickness cartilage loss  36 ± 15 95 ± 35  0 ± 0 Total degeneration width 436 ± 28 506 ± 57 343 ± 36Significant degeneration width 202 ± 13 236 ± 30 178 ± 17

In some embodiments, the compositions as disclosed herein provide for atherapeutic fusion protein that allows delivery and selective retentionof bioactive proteins at a desired site. HB-IGF-1 was retained inarticular cartilage and meniscus 4 to 8 days after injection at levelssufficient to stimulate proteoglycan synthesis. IGF-1 (no fused to HB)was not so retained. Accordingly, local delivery of HB-IGF-1 in vivo canreduce disease progression in a rat meniscal tear model of arthritis.Compared with IGF-1 or vehicle, HB-IGF-1 significantly reducedprogression of cartilage damage as measured by a modified OARSI score.Secondary analyses demonstrated a lower cartilage degeneration score andthe prevention of full-depth cartilage loss, suggesting a globalbeneficial effect on cartilage.

Although IGF-1 is one of the major anabolic growth factors forcartilage, attempts to repair cartilage and prevent osteoarthritis (OA)with intra-articular injection of IGF-1 alone have not been successful.Rogachefsky et al., 1993; Schmidt et al., 2006. The present datademonstrate that these negative results were not due to lack of effectof IGF-1 itself, but rather because “free” IGF-1 is not retained incartilage for a significant amount of time (IGF-1 was retained less than24 hours) after intraarticular delivery.

Additionally, these data have implications for injectable proteintherapies for cartilage in general. Development of future therapiesshould assess whether a targeting mechanism will be required to producesustained delivery to chondrocytes. The kinetics of retention incartilage can be verified in other models or experiments so that anegative experimental results of a particular agent is not interpretedas a failure of the agent itself Interestingly, FGF-18, another growthfactor that has been shown to be therapeutic in this model, is aheparin-binding growth factor ((Moore et al., 2005) Hu et al., 1998;Chuang et al., 2010)).

As demonstrated herein in the Examples, HB-PTH (but not PTH alone) hasalso been demonstrated to be retained in cartilage explants.

Further, the systemic pharmacokinetic data suggest that IGF levels werenot high enough to change glucose levels through binding to insulinreceptors. These results illustrate slowed systemic release of atherapeutic moiety after intra-articular injection.

In other embodiments, the HB-fusion protein comprises fibroblast growthfactor 18 (FGF-18) or a functional portion thereof. FGF family memberspossess broad mitogenic and cell survival activities, and are involvedin a variety of biological processes, including embryonic development,cell growth, morphogenesis, tissue repair. Thus, for example, the maturehuman FGF-18 can be incorporated into a HB-fusion protein, which FGF-18has the amino acid sequence:

EENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRSRRIRPTHPA (SEQ IDNO:31). See also Gene ID: 8817. HB can be fused to the N- or C- terminusof FGF-18 (e.g., HB-FGF, FGF-HB, HB-linker-FGF, or FGF-linker-HB) or aportion of FGF-18, for example, residues 1-169 of the mature FGF-18.

In some embodiments, the HB-fusion protein comprises parathyroid hormone(PTH) or a portion thereof. PTH is implicated in maintaining calciumlevels and osteostasis, and may prevent cartilage loss following jointinjury. See, e.g., Harrington et al., 290 Anatom. Rec. 155 (2007). Thus,for example, the PTH may be mature PTH, having the amino acid sequence:

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (SEQ ID NO:32).

In some embodiments, HB can be fused to the amino- or carboxy-terminusof PTH or a portion of PTH, such as amino acid residues 1-31, 1-34,1-37, 1-38, 1-44, or 1-84 of mature PTH. In some embodiments, a portionof PTH can be a fragment of the N-terminal 1-34 amino acids (referred toas PTH (1-34) corresponding to: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQID NO: 33).

In some embodiments, a portion of PTH fused to a HB peptide can beselected from the following amino acids sequences: PTH(1-31):SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV (SEQ ID NO: 34); PTH(1-37):SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVAL (SEQ ID NO: 35); PTH(1-44):SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPR (SEQ ID NO: 36).

As demonstrated herein in the Examples, HB-PTH(1-34) (but not PTH(1-34)alone) has also been demonstrated to be retained in cartilage explants.In some embodiments, a HB-PTH(1-34) fusion protein is a PTH(1-34)-linkerHB fusion protein and comprises amino acids:SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGG KRKKKGKGLGKKRDPRLRKYK (SEQ ID NO:37).

Additionally, the PTH may be an analog of PTH, such as cyclicPTH-(1-31). Recombinant human PTH(1-34) is currently marketed as FORTED(teriparatide [rDNA origin] injection) by Eli Lily & Co (Indianapolis,Ind.). OSTABOLIN-C, (ZT-031; cyclic PTH-(1-31)) has been investigated inclinical trials by Zelos Therapeutics, Inc. (West Conshohocken, Pa.).

In still other embodiments, the HB-fusion protein comprises PTHrP or aportion thereof. PTHrP has been implicated in chondroprotection of newlyregenerated cartilage following injury. Wang et al., 19 Osteoarth.Cartil. 213 (2011); Sampson et al., Ann. Meeting Am. Soc. Bone MineralRes. (2009). Mature PTHrP has the amino acids:

AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSPNTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTP (SEQ ID NO: 38). See also PLHLH, GeneID:5744. The PTHrP HB fusion protein may be arranged, for example:PTHrP-linker-HB; PTHrP(1-34)-linker-HB; PTHrP(1-36)-linker-HB;PTHrP(1-37)-linker-HB; PTHrP(1-40)-linker-HB, etc.

Another example of a PTHrP-HB fusion protein includes a syntheticportion of PTHrP

(AVSEHQLLHDKGKSIQDLRRRELLEKLLNKLHTA, where N is Aib (2-Aminoisobutyricacid) (SEQ ID NO: 39) can be fused to HB, for example,AVSEHQLLHDKGKSIQDLRRRELLEKLLNKLHTA(SEQ ID NO: 39)-linker-HB, where N isAib (2-Aminoisobutyric acid). Aib in this PTHrP analog or derivativedesignates α-aminoisobutyric acid (also called 2-Aminoisobutyric acid,α-methylalanine, or 2-methylalanine) This PTHrP-related polypeptide canbe made synthetically or by recombinant means, or by a combinationthereof. See Wang et al., 292 Science 498 (2001); Ryu & Schultz, 3 Nat.Methods 263 (2006). As noted, the compositions of the present inventioncan comprise HB-X2 or 2 or more different HB-X fusion proteins, etc.,where X represents two different active agents. Thus for example, acomposition can comprise any combination of HB-PTHrP and/or HB-PTH,and/or HB-IGF-1, e.g., for use in methods to regenerate cartilage, orstabilize regenerated cartilage following injury etc.

In yet another example, HB fusion protein fuses HB with IL 1 RA(Interleukin-1 receptor antagonist) as, for example, HB-linker-IL1RA orIL1RA-linker-HB. IL1RA is a member of the interleukin 1 cytokine family.IL1RA is secreted by various types of cells including immune cells,epithelial cells, and adipocytes, and is a natural inhibitor of thepro-inflammatory effect of IL1β. This protein inhibits the activities ofinterleukin 1, alpha (IL1A) and interleukin 1, beta (IL1β), andmodulates a variety of interleukin 1 related immune and inflammatoryresponses. See Arend, 13 Cytokine Growth Factor Rev. 323 (2002). In someembodiments, the HB is fused with IL-1/IL-1 RA chimeras (e.g., asdisclosed in Hou et al., PNAS, 2013, 110(10):3913-8), e.g., for use inthe treatment of damage or disease to cartilage and/or meniscus, or forthe treatment of eye or inflammatory conditions. Mature IL1RA has theamino acid sequence:RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO: 40). See also, Gene ID:3557.

Another embodiment of the present invention, the HB fusion comprises HBfused to HGMB2 (also called HMG2). Articular cartilage is a tissue thatprovides biomechanical properties that allow near frictionless jointmovement and dispersion of mechanical loads. Cartilage is composed of asingle cell lineage but differences in the organization, phenotype andfunction of cells in the various layers of cartilage have beenrecognized. The superficial zone (SZ) is the most unique. SZ cellsproduce lubricin, also termed proteoglycan-4 (PRG4) or superficial zoneprotein (SZP), an important joint lubricant; are more responsive tostimulation by catabolic cytokines such as IL-1 ; and expressmesenchymal stem cell markers. Expression of HMGB2 is restricted to theSZ of articular cartilage, and an interaction between HMGB2 and the Wnt/β-catenin pathway regulates the maintenance of the SZ and promoteschondrocyte survival. Importantly, joint ageing in humans and mice leadsto a loss of HMGB2 expression that correlated with the onset of OA-likechanges. Taniguchi et al., 106 PNAS 16817 (2009). Human HMG2 has theamino acid sequence (UniProtKB/Swiss-Prot: P26583.2; see also Gene ID:3148): MGKGDPNKPR GKMSSYAFFV QTCREEHKKK HPDSSVNFAE FSKKCSERWK TMSAKEKSKFEDMAKSDKAR YDREMKNYVP PKGDKKGKKK DPNAPKRPPS AFFLFCSEHR PKIKSEHPGLSIGDTAKKLG EMWSEQSAKD KQPYEQKAAK LKEKYEKDIA AYRAKGKSEA GKKGPGRPTGSKKKNEPEDE EEEEEEEDED EEEEDEDEE (SEQ ID NO: 83).

In some embodiments, a HB-fusion protein comprises somatostatin (SST) ora functional fragment or analogue thereof (e.g., a HB peptide can beconjugated to small molecules octreotide (brand name SANDOSTATIN®),pasireotide (SOM230, trade name SIGNIFOR®), lanreotide (trade name:SOMATULINE®)). Such a HB-fusion protein comprising SST or a functionalfragment or variant or analogue thereof can be used for the treatment ofdefects and disorders in cartilage and/or meniscus and anti-inflammatoryconditions. Human SST has the amino acid sequence (UniProtKB/Swiss-Prot:Hs.12409; see also Gene ID: 6750): MLSCRLQCAL AALSIVLALG CVTGAPSDPRLRQFLQKSLA AAAGKQELAK YFLAELLSEPNQTENDALEP EDLSQAAEQD EMRLELQRSANSNPAMAPRE RKAGCKNFFW KTFTSC (SEQ ID NO: 72).

In some embodiments, a HB-fusion protein comprises angiopoietin-like 3(ANGPTL3) or a functional fragment or analogue thereof. Such a HB-fusionprotein comprising SST or a functional fragment or variant or analoguethereof can be used for the treatment of defects and disorders incartilage and/or meniscus and anti-inflammatory conditions. HumanpreproproteinANGPTL3 has the amino acid sequence (UniProtKB/Swiss-Prot:Q9Y5C1; see also Gene ID: 27329 or NP 055310.1 ”):MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFV HKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNS KLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQ YKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE (SEQ ID NO: 73).

In some embodiments, a HB-fusion protein comprises a functional fragmentof angiopoietin-like 3 (ANGPTL3). Functional fragments of ANGPTL3include, but are not limited to:

Human ANGPTL3 (241-455) corresponding to:

GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD (SEQ ID NO: 74);

Human ANGPTL3 (225-455)TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD (SEQ ID NO: 75);

Human ANGPTL3 (207-455)IQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYS IKSTKMLIHPTD(SEQ ID NO: 76)

Brain proteoglycans, to which the HB associates, provide an avenue forcontrolled release of therapeutic agents to the central nervous system.See, e.g., Brandtlow & Zimmerman, 80 Physiol. Rev. 1267 (2000). Asdemonstrated herein in the Examples, a HB-IGF-1 fusion protein isretained in the spinal cord. According, in some embodiments, a HB-fusionprotein can comprise an active agent for delivery to the brain andspinal cord.

In some embodiments, a HB-fusion protein comprises IL-10. IL-10, ananti-inflammatory cytokine, is reportedly beneficial in subject havingmultiple sclerosis (MS); inducing anti-inflammatory cytokine IL-10provided clinical advantage in MS patients. Ersoy et al., 12 Eur. J.Neurol. 208 (2005). Hence, another embodiment provides for a HB-X fusionin which X is IL-10 or a portion thereof.

Alternatively, where X is a therapeutic protein portion of a recombinantHB-X fusion protein, X can be selected from Neurotrophic factors,including Neurotrophins such as nerve growth factor (NGF; Gene ID:4803), brain-derived neurotrophic factor (BDNF, Gene ID: 627),neurotrophin-3 (NT3, Gene ID:4908), neurotrophin-4 (NTF4, Gene ID:4909), Ciliary neurotrophic factor (CNTF, Gene ID: 1270), mesencephalicastrocyte-derived neurotrophic factor (MANF, Gene ID: 7873), or cerebraldopamine neurotrophic factor (CDNF, Gene ID: 441549); Glial cell-linederived neurotrophic factor family ligands such as glial cellline-derived neurotrophic factor (GDNF, Gene ID: 2668), neurturin (NRTN,Gene ID: 4902), artemin (ARTN, Gene ID: 9048), or persephin (PSPN, GeneID: 5623).

In some embodiments, the active agent X can be selected fromNeuropoietic cytokines such as interleukin-6 (IL6, Gene ID: 3569),interleukin-11 (IL1 1, Gene ID: 3589), interleukin-27 (IL27, Gene ID:246778), leukemia inhibitory factor (LIF, Gene ID: 3976), ciliaryneurotrophic factor (CNTF, Gene ID: 1270), cardiotrophin 1 (CTF1, GeneID: 1489), neuropoietin (NP ortholog of mouse, human pseudogene Gene ID:647088), cardiotrophin-like cytokine (CLGF1, Gene ID: 23529), orFibroblast Growth Factor 2 (FGF2, Gene ID: 2247);

In some embodiments, the active agent X can be selected fromAnti-inflammatory cytokines including interleukin-4 (ILR4, Gene ID:3565), and interleukin-10 (ILR10, Gene ID:3586);

In some embodiments, the active agent X can be selected fromNeuroprotection agents including Neuregulin-1 (NRG1, Gen ID: 3084), andVascular endothelial growth factor (VEGFA, Gene ID: 7422, VEGFB, GeneID: 7423, VEGFC, Gene ID: 7424).

Alternatively, in some embodiments the active agent X can be selectedfrom other therapeutic proteins such as CEREBROLYSIN® (FPF-1070 pigbrain peptide preparation, Ever Neuro Pharma, Austria), Growthdifferentiation factor 11 (GDF11, Gene ID: 10220), Stromal cell-derivedfactor-1 (SDF1, also CXCL12, Gene ID: 6387), Myostatin (MSTN, GeneID:2660), Parathyroid hormone (PTH, Gene ID: 5741); Parathyroid hormonerelated peptide (PTHrP or PLHLH, Gene ID: 5744); Interleukin 1 receptorantagonist (IL1RN, Gene ID: 3557); Fibroblast growth factor 18 (FGF18,Gene ID: 8817); High-mobility group box 2 (HMGB2, Gene ID: 3148); atherapeutic antibody or portion thereof, such as Remicade® (infliximab,anti-TNF-a, Janssen Biotech, Horsham, Pa.), Humira® (adalimumab,anti-TNF, Abbot Labs., N. Chicago, Ill.), or Enbrel® (etanercept,soluble recombinant TNF receptor 2 fused to the Fc component of humanimmunoglobulin G1, Amgen, Thousand Oaks, Calif.).

In some embodiments, the active agent X can be a Glucocorticoidreceptor, such as nuclear receptor subfamily 3, group C, member 1(NR3C1, Gene ID: 2908;

In some embodiments, the active agent X can be a portion, variant,analog, or derivative of any of preceding therapeutic proteins.

Active Agents—Therapeutic Small Molecules

In yet other embodiments, HB can be harnessed to effect controlledrelease of therapeutic small molecules. For example, the small moleculeTR2-01829, an optimized analog of PRO1, has chondrogenic properties.More specifically, PRO1 or an optimized analogue, a small moleculedevelopment candidate that has been shown to direct MSC differentiationdown the chondrogenic pathway, may prove useful in regenerative therapyfor OA. Additionally, 2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide(HS-Cf) was a potent inhibitor of NO production and iNOS expression inTNF-α-stimulated porcine chondrocytes. By down-regulating TNF-α-inducedIRF-1 activity, which suppressed chondrocyte activation and preventedcartilage destruction, HS-Cf might be a potential disease-modifying drugfor OA therapeutics. Liu et al., 31 J. Clin. Immunol. 1131 (2011). Invitro and mouse studies suggest the small molecule kartogenin could helptreat osteoarthritis. 5(18) SciBX (May 3, 2012).

The small molecule can be linked to the HB portion of the composition beany number of known approaches. Many bivalent or polyvalent linkingagents are useful in coupling protein molecules to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, disocyanates,glutaraldehydes, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. See Killen & Lindstrom, 133 J. Immunol. 1335 (1984);Jansen et al., 62 lmm. Rev. 185 (1982). In some embodiments,cross-linking reagents agents described in the literature areencompassed for use in the HB compositions as disclosed herein. See,e.g., Ramakrishnan, et al., 44 Cancer Res. 201 (1984) (describing theuse of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester)); Umemoto etal., U.S. Pat. No. 5,030,719 (describing the use of a halogenated acetylhydrazide derivative coupled to an antibody by way of an oligopeptidelinker). Particular linkers include: (a) EDC(1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (b) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (c) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat#21651G); (d) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (f) sulfo-NHS(N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkages or linking agents described above contain components thathave different attributes, thus leading to conjugates with differingphysio-chemical properties. For example, sulfo-NHS esters of alkylcarboxylates are more stable than sulfo-NHS esters of aromaticcarboxylates. NHS-ester containing linkers are less soluble thansulfo-NHS esters. Further, the linker SMPT contains a stericallyhindered disulfide bond, and can form conjugates with increasedstability. Disulfide linkages, are in general, less stable than otherlinkages because the disulfide linkage can be cleaved in vitro,resulting in less conjugate available. Sulfo-NHS, in particular, canenhance the stability of carbodimide couplings. Carbodimide couplings(such as EDC) when used in conjunction with sulfo-NHS, forms esters thatare more resistant to hydrolysis than the carbodimide coupling reactionalone.

Exemplary cross-linking molecules for use in the methods andcompositions as disclosed herein include, but are not limited to thoselisted in Tables 3 and 4:

TABLE 3 Exemplary homobifunctional crosslinkers (homobifunctionalcrosslinking reagents that have the same type of reactive group ateither end. Reagents are classified by what chemical groups they crosslink (left column) and their chemical composition (middle column).Products are listed in order of increasing length within each cell).Crosslinking Crosslinker Reactive Target Groups, Features ExampleProducts Amine-to-Amine NHS esters DSG; DSS; BS3; TSAT (trifunctional);Bioconjugate Toolkit Reagent Pairs NHS esters, PEG spacer BS(PEG)5;BS(PEG)9 NHS esters, thiol-cleavable DSP; DTSSP NHS esters,misc-cleavable DST; BSOCOES; EGS; Sulfo-EGS Imidoesters DMA; DMP; DMSImidoesters, thiol-cleavable DTBP Other DFDNB; THPP (trifunctional);Aldehyde-Activated Dextran Kit Sulfhydryl-to- Maleimides BMOE; BMB; BMH;Sulfhydryl TMEA (trifunctional) Maleimides, PEG spacer BM(PEG)2;BM(PEG)3 Maleimides, cleavable BMDB; DTME Pyridyldithiols (cleavable)DPDPB Other HBVS (vinylsulfone) Nonselective Aryl azides BASED(thiol-cleavable)

TABLE 4 Exemplary heterobifunctional crosslinkers (heterobifunctionalcrosslinking reagents that have the different reactive groups at eitherend. Reagents are classified by what chemical groups they cross link(left column) and their chemical composition (middle column). Productsare listed in order of increasing length within each cell.) CrosslinkingCrosslinker Reactive Targets Groups, Features Example Products Amine-to-NHS ester/Maleimide AMAS; BMPS; GMBS and Sulfhydryl Sulfo-GMBS; MBS andSulfo-MBS; SMCC and Sulfo-SMCC; EMCS and Sulfo-EMCS; SMPB andSulfo-SMPB; SMPH; LC-SMCC; Sulfo-KMUS NHS ester/Maleimide, SM(PEG)2;SM(PEG)4; PEG spacer SM(PEG)6; SM(PEG)8; SM(PEG)12; SM(PEG)24 NHS ester/SPDP; LC-SPDP and Pyridyldithiol, Sulfo-LC-SPDP; SMPT; cleavableSulfo-LC-SMPT NHS esters/Haloacetyl SIA; SBAP; SIAB; Sulfo-SIABAmine-to- NHS ester/Aryl Azide NHS-ASA Nonselective ANB-NOS Sulfo-HSABSulfo-NHS-LC-ASA SANPAH and Sulfo- SANPAH NHS ester/Aryl Azide,Sulfo-SFAD; Sulfo- cleavable SAND; Sulfo-SAED NHS ester/Diazirine SDAand Sulfo-SDA; LC-SDA and Sulfo- LC-SDA NHS ester/Diazirine, SDAD andSulfo-SDAD cleavable Amine-to-Carboxyl Carbodiimide DCC; EDCSulfhydryl-to- Pyridyldithiol/ APDP Nonselective Aryl AzideSulfhydryl-to- Maleimide/Hydrazide BMPH; EMCH; MPBH; Carbohydrate KMUHPyridyldithiol/ BMPH; EMCH; MPBH; Hydrazide KMUH Carbohydrate-to-Hydrazide/Aryl Azide ABH Nonselective Hydroxyl-to- Isocyanate/MaleimidePMPI Sulfhydryl Amine-to-DNA NHS ester/Psoralen SPB

The small molecules, and where relevant the fusion proteins, of the HB-Xcompositions can include pro-drugs. The term “pro-drug” refers to anycompound which releases an active parent drug in vivo when such pro-drugis administered to a mammalian subject. Pro-drugs of a compound aretypically prepared by modifying one or more functional group(s) presentin the compound in such a way that the modification(s) may be cleaved invivo to release the parent compound. Examples of pro-drugs include, butare not limited to, esters (e.g., acetate, formate, and benzoatederivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxyfunctional groups, and amides, carbamates and urea derivatives of aminofunctional groups, and the like. Pro-drug forms often offer advantagesof solubility, tissue compatibility, or delayed release in the mammalianorganism. See Bundgard, DESIGN PRODRUGS, 7, 21 (Elsevier, Amsterdam,1985); Silverman, ORGANIC CHEMISTRY DRUG DESIGN & DRUG ACTION, 352(Academic Press, San Diego, Calif.). Moreover, the prodrug derivativesof the invention may be combined with other features known to oneskilled in the art to enhance bioavailability.

HB-X Conjugates in Method of Treatment

Another aspect of the present invention relates to a method of treatinga cartilage-related condition (e.g., damage or disease) comprisingadministering to a subject an effective amount of a HB-X conjugate, suchas, for example, a recombinant fusion protein comprising HB-Xn or(HB-linker)n-Xn, wherein X is an active agent. In some embodiments, anactive agent for the treatment of a cartilage-related condition is atherapeutic protein. In some embodiments, therapeutic protein a selectedfrom Parathyroid hormone (PTH); Parathyroid hormone related peptide(PTHrP); Interleukin 1 receptor antagonist (IL-1RA); Fibroblast growthfactor 18 (FGF-18), an anti-nerve growth factor antibody; Fibroblastgrowth factor 9 (FGF-9); Hepatocyte growth factor; TGFβ-superfamilyproteins such as TGFβ, TGFβ3, BMP2, or BMP7; or portions, analogs,derivatives or functional fragments thereof.

In some embodiments, a HB-X conjugate for the treatment of acartilage-related condition can comprise at least one parathyroidhormone (PTH) therapeutic protein, e.g., one selected from any proteinor functional fragment thereof selected from the group of: SEQ ID NO:32-37.

In some embodiments, a HB-X conjugate for the treatment of acartilage-related condition can comprise at least one therapeuticprotein, e.g., one selected from any protein or functional fragmentthereof selected from the group of: IGF-1 or variants or functionalfragments thereof (SEQ ID NO: 6-9, SEQ ID NO: 63); FGF-18 (SEQ ID NO:31); PTH or variants or functional fragments thereof (SEQ ID NO: 32-37);PTHrP (SEQ ID NO: 38, 39); IL-1 RA or IL-1/IL-1 RA chimeras (SEQ ID NO:40); HMG (SEQ ID NO: 83); SST (SEQ ID NO: 72): ANGPTL3 or variants orfunctional fragments thereof (SEQ ID NO: 73-76); -41, SEQ ID NO: 63 andSEQ ID NO: 73-76, or functional fragments thereof.

In some embodiments, a HB-X conjugate for the treatment of acartilage-related condition can comprise at least one small molecule,e.g., but not limited to a SST agonist, e.g., octreotide, pasireotide,or lamreotide. In some embodiments, a HB-X conjugate as disclosed hereincomprising a IGF-1 protein or functional fragment or variant thereof(e.g., SEQ ID NO: 6-9 or SEQ ID NO: 63) can be used for the treatment ofa subject with dwarfism and/or a related condition with delayed growth.

Another aspect of the present invention relates to a method of treatinga cartilage-related condition (e.g., damage or disease) comprisingadministering to a subject an amount of a HB-X conjugate, such as, forexample, a recombinant fusion protein comprising HB-Xn or(HB-linker)n-Xn, where X is a Glucocorticoid receptor; and furtheradministering concurrently or separately a Corticosteroid.

In some embodiments, a cartilage-related condition is a articularcartilage defect including rupture or detachment, a meniscal defectincluding a partial or complete tear, Osteoarthritis, Traumaticcartilage rupture or detachment, disease or damage to the meniscusand/or patella, Ankylosing spondylitis, Capsulitis, Psoriatic arthritis,Rheumatoid arthritis, Systemic lupus erythematosus, Juvenile idiopathicarthritis, or X-linked hypophosphatemic rickets.

In some embodiments, a cartilage-related condition is a rupture ordetachment of the cartilage, a meniscal defect including a partial orcomplete tear or damage or a disease effecting the meniscus and/orpatella. In some embodiments, a cartilage-related condition is selectedfrom any or a combination of diseases from the following group:osteoarthritis (referred to herein as “OA” which results from breakdownof cartilage), including knee, finger, wrist, hip, ankle, elbow, toe,shoulder, and spinal osteoarthritis, traumatic cartilage rupture ordetachment, ankylosing spondylitis, capsulitis, psoriatic arthritis,rheumatoid arthritis (RA), systemic lupus erythematosus, juvenileidiopathic arthritis, Chondropathy, Chondrosarcoma, Chondromalacia,Polychondritis, Relapsing Polychondritis, Slipped epiphysis,Osteochondritis Dissecans, Chondrodysplasia, Costochondritis, X-linkedhypophosphatemic rickets, Osteochondroma, Chondrosarcoma (malignant),Osteoarthritis Susceptibility (types 1-6), Spondylosis, Osteochondroses,Primary chondrosarcoma, Chondrodysplasia, Tietze syndrome,Dermochondrocorneal dystrophy of Francois, Epiphyseal dysplasia,multiple, (types 1-5), Ossified Ear cartilages with Mental deficiency,Muscle Wasting and Bony Changes, Carpotarsal osteochondromatosis,Achondroplasia, Chondrocalcinosis (types 1-2), Genochondromatosis,Chondrodysplasia (disorder of sex development), Chondroma,Achondrogenesis (types 1A, 1B, 2, 3, 4, Langer-Saldino Type), Type IIAchondrogenesis-Hypochondrogenesis, Atelosteogenesis, (type 1, 2 andIII), Pyknoachondrogenesis, Pseudoachondroplasia, Osteoarthropathy offingers, familial, Diastrophic dysplasia, Dyschondrosteosis-nephritis,Coloboma of Alar-nasal cartilages with telecanthus, Alar cartilageshypoplasia-coloboma-telecanthus, Pierre Robin syndrome-fetalchondrodysplasia, Dysspondyloenchondromatosis, Achondroplasiaregional-dysplasia abdominal muscle, Osteochondritis Dissecans, FamilialArticular Chondrocalcinosis, Tracheobronchomalacia, Chondritis,Dyschondrosteosis, Maffucci Syndrome, Jequier-Kozlowski-skeletaldysplasia, Chondrodystrophy, Cranio osteoarthropathy, Tietze's syndrome,Hip dysplasia-enchondromata-enchondromata, Bessel-Hagen disease,Chondromatosis (benign), Enchondromatosis (benign), chondrocalcinosisdue to apatite crystal deposition, Meyenburg-Altherr-Uehlinger syndrome,Enchondromatosis-dwarfism-deafness, Astley-Kendall syndrome, Synovialosteochondromatosis, Chondrocalcinosis familial articular, Severeachondroplasia with developmental delay and acanthosis nigricans,Chondrocalcinosis, Keutel syndrome, Stanescu syndrome,Fibrochondrogenesis, Hypochondroplasia.

A subject amenable for the treatment with a HB-X conjugate for thetreatment of a cartilage-related condition is selected from a subjectwho has one or more symptoms of a joint disorder or cartilage loss ordamage, including one or more symptoms from the group of: jointswelling, joint pain, joint redness, joint laxity, mild arthritissymptoms, haemorrhagic joint effusion, inflammatory joint effusion,joint hypermobility, non-inflammatory joint effusion or other types.

In some embodiments, a subject is selected for administration of acomposition comprising a HB-X conjugate for the treatment of acartilage-related condition is a subject who has familialosteochondritis dissecans, where the subject has a mutation of the ACANgene. The ACAN gene provides instructions for making the aggrecanprotein, which is a component of cartilage. Aggrecan attaches to theother components of cartilage, organizing the network of molecules thatgives cartilage its strength. In addition, aggrecan attracts watermolecules and gives cartilage its gel-like structure. This featureenables the cartilage to resist compression, protecting bones andjoints. The ACAN gene mutation associated with familial osteochondritisdissecans results in an abnormal protein that is unable to attach to theother components of cartilage. As a result, the cartilage is abnormaland disorganized and weak and leads to the lesions and osteoarthritischaracteristic of familial osteochondritis dissecans.

In some embodiments, a subject is selected for administration of acomposition comprising a HB-X conjugate for the treatment of acartilage-related condition has an osteopenic related disease orosteoporosis, e.g., associated with the peri and post-menopausalconditions. Also encompassed are the treatment and prophylaxis ofPaget's disease, hypercalcemia associated with bone neoplasms and allthe types of osteoporotic diseases as classified below according totheir etiology: Primary osteoporosis, hypercalcemia, involutionalosteoporosis, Type I or postmenopausal osteoporosis, Type II or senileosteoporosis, Juvenile osteoporosis, Idiopathic in young adultsosteoporosis, Secondary osteoporosis, Endocrine abnormality,Hyperthyroidism, Hypogonadism, Ovarian agenesis, or Turner's syndrome,Hyperadrenocorticism or Cushing's syndrome, Hyperparathyroidism, Bonemarrow abnormalities, Multiple myeloma and related disorders, andSystemic mastocytosis, disseminated carcinoma osteoporosis, Gaucher'sdisease, Connective tissue abnormalities, Osteogenesis imperfecta,Homocystinuria, Ehlers-Danlos syndrome, Marfan's syndrome, Menke'ssyndrome, Miscellaneous causes Immobilisation or weightlessness,Sudeck's atrophy, chronic obstructive pulmonary disease, chronicalcoholism, chronic heparin administration and chronic ingestion ofanticonvulsant drugs

Patients amenable to treatment with a composition comprising a HB-Xconjugate for the treatment of a cartilage-related condition asdisclosed herein include patients at risk of disease but not showingsymptoms (for example asymptomatic patients), as well as patientspresently showing symptoms. In the case of OA or osteoporosis, virtuallyanyone, particularly women are at risk of suffering from OA andosteoporosis if he or she lives long enough.

In some embodiments, a subject is selected for administration of acomposition comprising a HB-X conjugate for the treatment of acartilage-related condition is a subject known to have a genetic risk ofa cartilage-related disease or disorder, e.g., OA. In some embodiments,patients are women, for example post-menopausal, or women at least 65years of age, or patients who have had previous fractures or haverelatives who have had a metabolic bone disease, for exampleosteoporosis. Patients can be identified as having increased risk ofdeveloping metabolic bone disease using methods commonly known by personof ordinary skill in the art.

In some embodiments, a subject is selected for administration of acomposition comprising a HB-X conjugate for the treatment of acartilage-related condition has at least one of the followingconditions; rheumatoid arthritis (RA), Juvenile Rheumatoid Arthritis(JRA), psoriatic arthritis, Reiter's syndrome (reactive arthritis),Crohn's disease, ulcerative colitis and sarcoidosis (Orcel, et al., Bonedemineralization and cytokines; Rev Rhum Mal Osteoartic. 1992; 59:16S-22S; Brown, et al., The radiology of rheumatoid arthritis. Am FamPhysician. 1995. 52: 1372-80; De Vos, et al., Bone and joint diseases ininflammatory bowel disease. Aliment Pharmacol Ther. 1998; 12(5):397-404;Falcini, et al., The primary role of steroids on the osteoporosis injuvenile rheumatoid patients evaluated by dual energy X-rayabsorptiometry. J Endocrinol Invest. 1996; 19(3): 165-9; Scutellari, etal., Rheumatoid arthritis: sequences. Eur J. Radiol. 1998: Suppl1:S31-8).

Rheumatoid arthritis is associated with a decrease in bone mass (Cortet,et al., Evaluation of bone mineral density in patients with rheumatoidarthritis. Influence of disease activity and glucocorticoid therapy. RevRhum Engl Ed. 1997 July-Sep. 30, 1997; 64(7-9):451-8). Typical changesof an inflammatory arthritis include juxta-articular osteoporosis,cartilage loss, and cortical or marginal bone erosions (Lawson, et al.,Lyme arthritis: radiologic findings. Radiology. 1985; 154(1):37-43;Grassi, et al., The clinical features of rheumatoid arthritis. Eur J.Radiol. 1998; 1:S18-24).

In some embodiments, a subject is selected for administration of acomposition comprising a HB-X conjugate for the treatment of acartilage-related condition who has a chronic inflammatory jointdisease, such as rheumatoid arthritis, synovial cells produce largeamounts of cytokines leading to increased local bone resorption andjuxta-articular bone destructions (Orcel, et al., 1992, Bonedemineralization and cytokines Rev Rhum Mal Osteoartic 59(6 Pt2):165-22S).

Another aspect of the present invention relates to a method of treatinga neurological condition (e.g., a disorder or disease) comprisingadministering to a subject an amount of a HB-X conjugate, such as, forexample, a recombinant fusion protein comprising HB-Xn or(HB-linker)n-Xn, where X is a therapeutic protein selected from nervegrowth factor (NGF), brain-derived neurotrophic factor (BDNF),neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), Ciliary neurotrophicfactor (CNTF), mesencephalic astrocyte-derived neurotrophic factor(MANF), conserved dopamine neurotrophic factor (CDNF), glial cellline-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin(ARTN), persephin (PSPN), interleukin-6, interleukin-11, interleukin-27,leukaemia inhibitory factor, ciliary neurotrophic factor, cardiotrophin1, neuropoietin, cardiotrophin-like cytokine, FPF-1070, FibroblastGrowth Factor 2, Neuregulin-1, Vascular endothelial growth factor(VEGF), or a functional portion, analog, or derivative thereof.

In some embodiments, a neurological condition to be treated is selectedfrom the group of neurological diseases selected from Alzheimer'sdisease, Parkinson's disease, Amyotrophic lateral sclerosis, Multiplesclerosis, Brain injury, Spinal cord injury, Peripheral nervedegeneration, Stroke, Huntington's disease, Pick's disease, Diabeticneuropathy, Frontotemporal dementia, Dementia with Lewy bodies,Corticobasal degeneration, Progressive supranuclear palsy, Priondisorders, Progressive supranuclear palsy, Multiple system atrophy,Hereditary spastic paraparesis, Spinocerebellar atrophies, Friedreich'sataxia, Amyloidoses, or Charcot Marie Tooth syndrome.

Accordingly, in some embodiments, a subject is selected foradministration of a composition comprising a HB-X conjugate for thetreatment of a neurological disease or disorder who has at least one ofthe following diseases and disorders, including, but not limited toAlzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease(HD), vascular dementia, aging and mild-cognitive impairment. In someembodiments, a subject selected for treatment has a neurodegenerativedisorder is selected from the group consisting of: Alzheimer's diseaseand Parkinson disease.

In some embodiments, a subject is selected for treatment who has aneurological disorder such as: hyperosmolarity; acidic pH; burnencephalopathy; lead encephalopathy; autoimmune encephalitis; multiplesclerosis; post-ischemia reperfusion; acute hypertension; microwaveirradiation; hepatic encephalopathy; seizures; tumors; development;hypervolemia; hypothermia; post-radiation; hyperbaric conditions;meningitis; lymphostatic encephalopathy; Wernickes-Korsakoff syndrome;familial mental retardation and amyotrophic lateral sclerosis (ALS).

Subjects amenable to treatment with a composition comprising a HB-Xconjugate for the treatment of a neurological disease or disorder asdisclosed herein include subjects at risk of disease but not showingsymptoms (for example asymptomatic subjects), as well as subjectspresently showing symptoms. In the case of Alzheimer's disease,virtually anyone is at risk of suffering from Alzheimer's disease if heor she lives long enough. Therefore, the present methods can beadministered prophylactically to the general population without anyassessment of the risk of the subject patient. The methods as disclosedherein are especially useful for individuals who do have a known geneticrisk of Alzheimer's disease. Such individuals include those havingrelatives who have experienced this disease, and those whose risk isdetermined by analysis of genetic or biochemical markers, as disclosedherein Alzheimer's disease (AD) is a progressive disease resulting insenile dementia and generally falls into two categories: late onset,which occurs in old age (65+ years) and early onset, which develops wellbefore the senile period, i.e., between 35 and 60 years. In both typesof disease, the pathology is the same but the 0 abnormalities tend to bemore severe and widespread in cases beginning at an earlier age. Thedisease is characterized at the macroscopic level by significant brainshrinkage away from the cranial vault as seen in MRI images as a directresult of neuronal loss and by two types of macroscopic lesions in thebrain, senile plaques and neurofibrillary tangles. Senile plaques areareas comprising disorganized neuronal processes up to 150 μm across andextracellular amyloid deposits, which are typically concentrated at thecenter and visible by microscopic analysis of sections of brain tissue.Neurofibrillary tangles are intracellular deposits of tau proteinconsisting of two filaments twisted about each other in pairs.

Genetic markers of risk toward Alzheimer's disease include mutations inthe APP gene, particularly mutations at position 717 and positions 670and 671 referred to as the Hardy and Swedish mutations respectively (seeHardy, TINS, supra). Other markers of risk are mutations in thepresenilin genes, PS1 and PS2, and ApoE4, family history of Alzheimer'sdisease, hypercholesterolemia or atherosclerosis. Subjects presentlysuffering from Alzheimer's disease can be recognized from characteristicdementia, as well as the presence of risk factors described above. Inaddition, a number of diagnostic tests are available for identifyingsubjects who have Alzheimer's disease. These include measurement of CSFtau and Aβ42 levels. Elevated tau and increased Aβ42 levels signify thepresence of Alzheimer's disease. Individuals suffering from Alzheimer'sdisease can also be diagnosed by MMSE or ADRDA criteria. The tissuesample for analysis is typically blood, plasma, serum, mucus or cerebralspinal fluid from the patient. The sample is analyzed for indicia of animmune response to any forms of Aβ peptide, typically Aβ42. The immuneresponse can be determined from the presence of, e.g., antibodies orT-cells that specifically bind to Aβ peptide. ELISA methods of detectingantibodies specific to Aβ are commonly known to one of ordinary ski; inthe art.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingpresence of Aβ peptide in the CSF. If the Aβ peptide is still present inthe CSF, additional treatment with a HB-X conjugate for aneurodegenerative disease as disclosed herein are recommended, and/ortreatment of additional therapies for Alzheimer's disease. In the caseof potential Down's syndrome patients, treatment can begin antenatallyby administering therapeutic agent to the mother or shortly after birth.

In some embodiments, a composition comprising a HB-X conjugate for thetreatment of a neurological disease or disorder as disclosed herein arealso useful in the treatment of other neurodegenerative disorders orcognitive impairment disorders in general: for example, dementia,depression, confusion, Creutzfeldt-Jakob or mad cow disease,Huntington's disease, loss of motor coordination, multiple sclerosis,Parkinson's disease, Pick disease and other brain storage disorders(e.g., amyloidosis, gangliosidosis, lipid storage disorders,mucopolysaccharidosis), syncope, and vascular dementia. Thus, treatmentcan be directed to a subject who is affected with asymptomatic by theneurodegenerative disease; it can improve cognitive function.

Another aspect of the present invention relates to a method of treatingan eye disorder or disease comprising administering to a subject anamount of a HB-X conjugate, such as, for example, a recombinant fusionprotein comprising HB-Xn or (HB-linker)n-Xn, where X is a therapeuticprotein selected from nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4(NT-4), Ciliary neurotrophic factor (CNTF), mesencephalicastrocyte-derived neurotrophic factor (MANF), conserved dopamineneurotrophic factor (CDNF), glial cell line-derived neurotrophic factor(GDNF), neurturin (NRTN), artemin (ARTN), persephin (PSPN),interleukin-6, interleukin-11, interleukin-27, leukaemia inhibitoryfactor, ciliary neurotrophic factor, cardiotrophin 1, neuropoietin,cardiotrophin-like cytokine, FPF-1070, Fibroblast Growth Factor 2,Neuregulin-1, Vascular endothelial growth factor (VEGF), or a functionalportion, analog, or derivative thereof.

In some embodiments, a subject is selected for treatment with acomposition comprising HB-X for the treatment of an eye disease ordisorder has one or more eye disease from the following: Cornealulcer/Corneal abrasion, Thygeson's superficial punctate keratopathy,Corneal neovascularization, Fuchs' dystrophy, Keratoconjunctivitissicca, Chorioretinal inflammation, Chorioretinal scars, Choroidaldegeneration, Hereditary choroidal dystrophy, Retinal detachment,Retinoschisis, Hypertensive retinopathy, Retinopathy of prematurity,Age-related macular degeneration (AMD), Retinal degeneration, Maculardegeneration, Epiretinal membrane, Peripheral retinal degeneration,Hereditary retinal dystrophy, Retinitis pigmentosa, Xerophthalmia, orRetinal haemorrhage.

In some embodiments, a subject is selected for treatment with acomposition comprising HB-X for the treatment of an eye disease ordisorder has one or more eye disease from the following, but not limitedto diabetic retinopathy, retinopathy of prematurity (ROP), age-relatedmacular degeneration (AMD), retinal vein occlusion, radiationretinopathy. In some embodiments, a subject is selected for treatmentwith a composition comprising HB-X for the treatment of an eye diseaseor disorder who has an “inflammation-mediated condition of the eye”,which refers to herein as any condition of the eye which may benefitfrom treatment with an anti-inflammatory agent, and is meant to include,but is not limited to, uveitis, macular edema, acute maculardegeneration, retinal detachment, ocular tumors, fungal or viralinfections, multifocal choroiditis, diabetic uveitis, proliferativevitreoretinopathy (PVR), sympathetic opthalmia, Vogt Koyanagi-Harada(VKH) syndrome, histoplasmosis, and uveal diffusion.

Another aspect of the present invention relates to a method of treatinginflammation in a subject comprising administering to a subject anamount of a HB-X conjugate, such as, for example, a recombinant fusionprotein comprising HB-Xn or (HB-linker)n-Xn, where X is a therapeuticprotein or a portion thereof, selected from TNF receptor 2,interleukin-4, or interleukin-10. In some embodiments, a compositioncomprising HB-X for the treatment of inflammation comprises a cytokine(e.g., an anti-inflammatory cytokine) or chemokine as an active agent.

As used herein, a “cytokine” is a generic term for proteins released byany of the lymph cells that act on other cells as intercellularmediators and affect cellular activity and control inflammation.Cytokines are typically soluble proteins or peptides which are naturallyproduced by mammalian cells and which act in vivo as humoral regulatorsat micro- to picomolar concentrations. Cytokines can, either undernormal or pathological conditions, modulate the functional activities ofindividual cells and tissues. An anti-inflammatory cytokines, such asIL-4, IL-10, IL-11, W-13, IL-13 and TGFβ, are not mediators ofinflammation. Additionally examples of cytokines include, lymphokines,monokines, and traditional polypeptide hormones. Included among thecytokines are growth hormones such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone;thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoproteinhormones such as follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), and luteinizing hormone (LH); hepatic growth factor;fibroblast growth factor; prolactin; placental lactogen; tumor necrosisfactor-α and -β; mullerian-inhibiting substance (MIS); mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor (VEGF); integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-β; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as, for example and notfor limitation, IL-1, IL-11α., IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including leukemiainhibitory factor (LIF) and kit ligand (KL).

The term “chemokine” is a generic term for proteins that act on whiteblood cells and induce them to move and/or become activated to carry outtheir immune system functions. Chemokines are well-known in the art.Exemplary chemokines include, for example and not for limitation, TECK,ELC, BLC-1, CTACK, RANTES, fractalkine, exotaxin, eotaxin-2, Monocytechemoattractant protein-1 (MCP-1), MCP-2, MCP-3, MCP-4, MDC,leukotactin, SDF-1β., lymphotactin, TARC, ITAC, ENA-70, ENA-78, IP-10,NAP-2, interleukin-8 (IL-8), HCC-1, MIP-1α, MIP-1β, MIP-1δ, I-309,GRO-α, GRO-β, GRO-γ, MPIF-1, I-LINK, and GCP-2.

In some embodiments, a composition for the treatment of a subject withdwarfism and/or a related condition with delayed growth comprises anactive agent which is a IGF-1 protein or functional fragment or variantthereof (e.g., SEQ ID NO: 6-9 or SEQ ID NO: 63).

In some embodiments, a composition comprising HB-X for the treatment ofinflammation in a subject comprises an active agent which is a steroidalanti-inflammatory agent. Preferably, the steroidal anti-inflammatoryagent is selected from the group consisting of 21 acetoxypregnenolone,alclometasone, algestone, amcinonide, beclomethasone, betamethasone,budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone,cloprednol, corticosterone, cortisone, cortivazol, deflazacort,desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone,difluprednate, enoxolone, fluazacort, flucloronide, flumethasone,flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl,fluocortolone, fluorometholone, fluperolone acetate, fluprednideneacetate, fluprednisolone, flurandrenolide, fluticasone propionate,formocortal, halcinonide, halobetasol propionate, halometasone,halopredone acetate, hydrocortamate, hydrocortisone, loteprednoletabonate, mazipredone, medrysone, meprednisone, methylprednisolone,mometasone furoate, paramethasone, prednicarbate, prednisolone,prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate,prednisone, prednival, prednylidene, rimexolone, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide, andtriamcinolone hexacetonide. In a preferred embodiment, the steroidalanti-inflammatory agent is selected from the group consisting ofcortisone, dexamethasone, hydrocortisone, methylprednisolone,prednisolone, prednisone, and triamcinolone. In a more preferredembodiment, the steroidal anti-inflammatory agent is dexamethasone. Inanother embodiment, the bioerodible implant comprises more than onesteroidal anti-inflammatory agent.

In some embodiments, a composition comprising HB-X for the treatment ofinflammation comprises an anti-inflammatory agent. Preferably theanti-inflammatory agent is selected from the group consisting of: ananalgesic; an antirheumatic agent; an gastrointestinal agent; a goutpreparation; glucocorticoids; opthalmic preparation; respiratory agent;a nasal preparation; and a mucous membrane agent.

In some embodiments, a subject is selected for treatment with acomposition comprising HB-X for the treatment of an inflammation or aninflammatory-related disease or disorder has one or more of theinflammatory-related diseases from the following: arthritis, rheumatoidarthritis, an inflammatory bowel disease; psoriasis; multiple sclerosis;a neurodegenerative disorder; congestive heart failure; stroke; aorticvalve stenosis; kidney failure; lupus; pancreatitis; allergy; fibrosis;anemia; atherosclerosis; a metabolic disease; a bone disease; acardiovascular disease, a chemotherapy/radiation related complication;diabetes type I; diabetes type II; a liver disease; a gastrointestinaldisorder; an ophthalmological disease; allergic conjunctivitis; diabeticretinopathy; Sjogren's syndrome; uvetitis; a pulmonary disorder, a renaldisease; dermatitis; HIV-related cachexia; cerebral malaria; ankylosingspondolytis; leprosy; anemia; and fibromyalgia.

In some embodiments, a subject is selected for treatment with acomposition comprising HB-X for the treatment of an inflammation or aninflammatory-related disease has inflammatory bowel disease (IBD),specifically including Crohn's disease and ulcerative colitis. Inanother embodiment the disease being treated is arthritis, rheumatoidarthritis, psoriasis, Alzheimer's disease, or Parkinson disease. In yetanother preferred embodiment the disease is post-radiotherapy relateddisease or atherosclerosis.

In some embodiments, a subject is selected for treatment with acomposition comprising HB-X for the treatment of an inflammation or aninflammatory-related disease has inflammatory bowel disease selectedfrom the group consisting of: Crohn's disease or ulcerative colitis; agastrointestinal complication such as diarrhea; a liver disease isselected from the group consisting of: an autoimmune hepatitis,hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis,or fulminant liver failure; a gastrointestinal disorder selected fromthe group consisting of: celiac disease and non-specific colitis; a bonedisease is osteoporosis; the pulmonary disorder is selected from thegroup consisting of: allergic rihinitis, asthma, chronic obstructivepulmonary disease, chronic granulomatous inflammation, cystic fibrosis,and sarcoidosis; a cardiovascular disease selected from the groupconsisting of: atherosclerotic cardiac disease, congestive heart failureand restenosis; and a renal disease selected from the group consistingof: glomerulonephritis and vasculitis.

In some embodiments, a subject selected for treatment with a compositioncomprising HB-X for the treatment of inflammation has an auto-immunedisease. In some embodiments, a subject to be treated with a compositioncomprising HB-X for the treatment of inflammation or auto-immune diseasehas one or more of the following conditions from the following:rheumatoid arthritis, multiple sclerosis (MS), systemic lupuserythematosus (SLE), autoimmune myocarditis, sepsis, Graves' disease(overactive thyroid), Hashimoto's thyroiditis (underactive thyroid),Type 1 diabetes mellitus, celiac disease, Crohn's disease and ulcerativecolitis, Guillain-Barre syndrome, primary biliary sclerosis/cirrhosis,sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon,scleroderma, Sjogren's syndrome, Goodpasture's syndrome, Wegener'sgranulomatosis, polymyalgia rheumatica, temporal arteritis/giant cellarteritis, chronic fatigue syndrome CFS), psoriasis, autoimmuneAddison's Disease, ankylosing spondylitis, Acute disseminatedencephalomyelitis, antiphospholipid antibody syndrome, aplastic anemia,idiopathic thrombocytopenic purpura, Myasthenia gravis, opsoclonusmyoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus,pernicious anaemia, polyarthritis in dogs, Reiter's syndrome, Takayasu'sarteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosisand fibromyalgia (FM). Chronic inflammation recently has receivedinterest as a suspected cause and/or as a contributory factor in avariety of disease conditions. Perhaps most prominent among suchconditions are cardiovascular diseases, although cancers, similarly, areoften viewed as being developmentally related to chronic inflammation.

Administration of Pharmaceutical Compositions

An effective amount, e.g., a therapeutically effective dose of an HB-Xconjugate comprising a therapeutic protein or peptide may beadministered to the patient in a single dose or in multiple doses. Whenmultiple doses are administered, the doses may be separated from oneanother by, for example, one hour, three hours, six hours, eight hours,one day, two days, one week, two weeks, or one month. For example, acomposition comprising HB-X can be administered for, e.g., 2, 3, 4, 5,6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that, for anyparticular subject, specific dosage regimes should be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions. For example, the dosage of the therapeutic can beincreased if the lower dose does not provide sufficient therapeuticactivity. While the attending physician ultimately will decide theappropriate amount and dosage regimen, an effective amounts of a HB-Xcan provided at a dose of 0.0001, 0.01, 0.01 0.1, 1, 5, 10, 25, 50, 100,500, or 1,000 mg/kg. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model testbioassays or systems.

Dosages for a particular patient or subject can be determined by one ofordinary skill in the art using conventional considerations, (e.g. bymeans of an appropriate, conventional pharmacological protocol). Aphysician may, for example, prescribe a relatively low dose at first,subsequently increasing the dose until an appropriate response isobtained. The dose administered to a patient is sufficient to effect abeneficial therapeutic response in the patient over time, or, e.g., toreduce symptoms, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularformulation, and the activity, stability or serum half-life of theactive agent X e.g., the therapeutic peptide or protein as disclosedherein, and the condition of the patient, the disease to be treated, aswell as the body weight or surface area of the patient to be treated.The size of the dose is also determined by the existence, nature, andextent of any adverse side-effects that accompany the administration ofa particular vector, formulation, or the like in a particular subject.Therapeutic compositions comprising HB-X thereof are optionally testedin one or more appropriate in vitro and/or in vivo animal models ofdisease, such a cartilage assay as disclosed herein, or other modelscommonly known to persons of ordinary skill in the art, to confirmefficacy, tissue metabolism, retention in the tissue over time, systemichalf-life, speed of degradation, and to estimate dosages, according tomethods well known in the art. In particular, dosages can be initiallydetermined by activity, stability or other suitable measures oftreatment vs. non-treatment (e.g., comparison of treated vs. untreatedcells or animal models), in a relevant assay.

Formulations are administered at a rate determined by the LD50 of therelevant formulation, and/or observation of any side-effects of HB-X atvarious concentrations, e.g., as applied to the mass and overall healthof the patient. Administration can be accomplished via single or divideddoses.

In determining the effective amount of HB-X to be administered in thetreatment or prophylaxis of a disease, the physician evaluatescirculating plasma levels, formulation toxicities, and progression ofthe disease. The selected dosage level will also depend upon a varietyof factors including the activity of the particular compound of thepresent invention employed, or the ester, salt or amide thereof, theroute of administration, the time of administration, the rate ofexcretion of the particular compound being employed, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith the particular compound employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

In some embodiments, HB-X as disclosed herein can be administered at adose in accordance with good medical practice, taking into account theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Dosage regimensof a composition comprising HB-X as disclosed herein can be adjusted toprovide the optimum desired response (e.g. a therapeutic or prophylacticresponse). For example, a single bolus can be administered, severaldivided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage.

Furthermore, actual dosage levels of HB-X in a pharmaceuticalcomposition can be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular subject, composition, and mode ofadministration, without being toxic to the subject. A pharmaceuticalcomposition comprising HB-X as disclosed herein can be a“therapeutically effective amount” and/or a “prophylactically effectiveamount”. In general, a suitable daily dose of a composition comprisingHB-X as disclosed herein will be that amount of the active agent X whichis the lowest dose effective to produce a therapeutic effect, such as areduction of a symptom of a disease or an elicitation of an immune orallergenic response. Such an effective dose will generally depend uponthe factors described above.

If desired, the effective daily dose of a composition comprising HB-Xcan be administered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

It is to be noted that dosage values may vary with the type and severityof the disease to be alleviated. It is to be further understood that forany particular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

The efficacy and toxicity of the compound can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose is effective in 50% of the population) and LD50(the dose is lethal to 50% of the population). The dose ratio of toxicto therapeutic effects is the therapeutic index, and it can be expressedas the ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. For example, a therapeuticallyeffective amount can be estimated initially either in cell cultureassays or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model is also used to achieve a desirable concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in other subjects. Generally,the therapeutically effective amount is dependent of the desiredtherapeutic effect. For example, the therapeutically effective amount ofHB-X for the treatment of a cartilage-related disease or disorder onecan be assess the effect of the HB-X, e.g., HB-IGF-1 in an in vivo in arat model after transection of the medial meniscus (e.g., medialmeniscal tear (MMT) surgery) as disclosed herein in the Examples, whichis a rat model of surgically induced OA. After injection of the HB-Xconjugate (e.g. HB-IGF-1) histological assessment of knee osteoarthritis(OA) is performed and overall OARSI score is determined for the jointsof the animals treated with the HB-X conjugate (e.g., HB-IGF-1) iscompared to control treated animals, as described herein in theExamples.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. It is also noted thathumans are treated generally longer than the mice or other experimentalanimals exemplified herein, which treatment has a length proportional tothe length of the disease process and drug effectiveness. The doses maybe single doses or multiple doses over a period of several days, butsingle doses are preferred.

In some embodiments, HB-X conjugate can be administered to humans andother animals for therapy by any suitable route of administration,including orally, nasally, as by, for example, a spray, rectally,intravaginally, by intra-articular injection, parenterally,intracisternally and topically, as by powders, ointments or drops,including buccally and sublingually.

An HB-X conjugate can be administered by any route known in the art ordescribed herein, for example, oral, parenteral (e.g., intravenously orintramuscularly), intraperitoneal, rectal, cutaneous, nasal, vaginal,inhalant, skin (patch), or ocular. The HB-X conjugate may beadministered in any dose or dosing regimen.

In some embodiments, a HB-X conjugate composition as disclosed hereinwere the X is a immunogenic entity or an allergenic entity can beformulated to be suitable for intramuscular, intranasal, oral,subcutaneous, or intraperitoneal administration. Such formulationstypically comprise sterile aqueous solutions of the active ingredientwith solutions which are preferably isotonic with the blood of therecipient. Such formulations may be conveniently prepared by dissolvingsolid active ingredient in water containing physiologically compatiblesubstances such as sodium chloride (e.g., 0.1-2.0 M), glycine, and thelike, and having a buffered pH compatible with physiological conditionsto produce an aqueous solution, and rendering the solution sterile.These may be present in unit or multi-dose containers, for example,sealed ampoules or vials.

Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811, which is incorporated herein in its entirety by reference.Formulations for an intranasal delivery are described in U.S. Pat. Nos.5,427,782, 5,843,451 and 6,398,774, which are incorporated herein intheir entirety by reference.

In some embodiments, the formulations of the compositions comprising aHB linked or fused to an immunogenic entity or an allergenic entity canincorporate a stabilizer. Illustrative stabilizers are polyethyleneglycol, proteins, saccharide, amino acids, inorganic acids, and organicacids which may be used either on their own or as admixtures. Two ormore stabilizers may be used in aqueous solutions at the appropriateconcentration and/or pH. The specific osmotic pressure in such aqueoussolution is generally in the range of 0.1-3.0 osmoses, preferably in therange of 0.80-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8. In someembodiments, when oral preparations are desired, the compositions can becombined with typical carriers, such as lactose, sucrose, starch, talcmagnesium stearate, crystalline cellulose, methyl cellulose,carboxymethyl cellulose, glycerin, sodium alginate or gum arabic amongothers.

A method of immunization or injecting a mammal to induce an immuneresponse to an immunogenic entity fused to a HB peptide comprisesadministering a vaccine composition as described herein. In someembodiments, administration of the composition as a vaccine can beconducted by conventional methods. For example, at least one HB fused toan immunogenic entity can be used in a suitable diluent such as salineor water, or complete or incomplete adjuvants. The composition can beadministered by any route appropriate for eliciting an immune response.The composition can be administered once or at periodic intervals untilan immune response is elicited. Immune responses can be detected by avariety of methods known to those skilled in the art, including but notlimited to, antibody production, cytotoxicity assay, proliferation assayand cytokine release assays. For example, samples of blood can be drawnfrom the immunized mammal, and analyzed for the presence of antibodiesagainst the antigen proteins by ELISA (see de Boer G F, et. al., 1990,Arch Virol. 115:47-61) and the titer of these antibodies can bedetermined by methods known in the art. The precise dose to be employedin the formulation will also depend on the route of administration andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. For example, a range of 25 μg-900 μg totalprotein can be administered intradermally, monthly for about 3 months ormore. Ultimately, the attending physician will decide the amount ofprotein or composition to administer to particular individuals.

In some embodiments, a HB-X conjugate can be administered to a subjectas part of a biological implant or transplant. In some embodiments, abiological implant or transplant is incubated with a HB-X conjugate asdisclosed herein for a period of time prior to implanting the implant ortransplant into the subject. Any biological implant known to one ofordinary skill in the art are encompassed for use herein, for example,but not limited to, osteochondrial or meniscal allografts. In someembodiments, a biological scaffold is incubated with a HB-X conjugate asdisclosed herein for a period of time prior to implanting the scaffoldinto the subject. In some embodiments, the scaffold is a biocompatibleand/or biodegradable scaffold.

In some embodiments, a HB-X conjugate is administered to the subject ina hydrogel composition. Any biologically compatible hydrogel compositioncan be used, e.g., for example, but not limited to, a hydrogelcomprising self-assembling peptides. In some embodiments, a hydrogelcomprising self-assembling peptides is RADA-16 (also known asPURAMATRIX®) and KLD-12. Self-assembly peptide hydrogels are known toone of ordinary skill in the art, such as those described in US Pub.2013/0129712 and U.S. Pub. 2013/0079421, each incorporated by reference.

In some embodiments, a hydrogel may comprise peptides with the sequence

RADARADARADARADA (SEQ ID NO: 77) and/or the sequence KLDLKLDLKLDL (SEQID NO: 78). In some embodiments, the hydrogel comprisesAcN-KLDLKLDLKLDL-CNH2 (SEQ ID NO: 79). Accordingly, in some aspects ofthe present invention relate to a method for administering a HB-Xconjugate to a subject in need thereof, wherein the HB-X conjugate ispresent in a hydrogel comprising peptides selected from the group of:RADARADARADARADA (SEQ ID NO: 77), KLDLKLDLKLDL (SEQ ID NO: 78) orAcN-KLDLKLDLKLDL-CNH2 (SEQ ID NO: 79). In some embodiments, the presentinvention relates to a method of treating cartilage-related ormeniscus-related clinical condition comprising administering acomposition comprising a HB-X and a hydrogel, e.g., a hydrogelcomprising peptides selected from the group of: RADARADARADARADA (SEQ IDNO: 77), KLDLKLDLKLDL (SEQ ID NO: 78) or AcN-KLDLKLDLKLDL-CNH2 (SEQ IDNO: 79). In some embodiments, the present invention relates to a methodof treating a neuronal disease or disorder comprising administering acomposition comprising a HB-X and a hydrogel, e.g., a hydrogelcomprising peptides selected from the group of: RADARADARADARADA (SEQ IDNO: 77), KLDLKLDLKLDL (SEQ ID NO: 78) or AcN-KLDLKLDLKLDL-CNH2 (SEQ IDNO: 79).

When the agents or compounds are delivered to a subject, they can beadministered by any suitable route, including, for example, orally(e.g., in capsules, suspensions or tablets) or by parenteraladministration. Parenteral administration can include, for example,intramuscular, intravenous, intraarticular, intra-arterial, intrathecal,intradermal, subcutaneous, or intraperitoneal administration. The agentcan also be administered orally, transdermally, topically, by inhalation(e.g., intra-bronchial, intranasal, oral inhalation or intranasal drops)or rectally. Administration can be local or systemic as indicated.Agents can also be delivered using viral vectors, which are well-knownto those skilled in the art. The pharmaceutically acceptableformulations can be suspended in aqueous vehicles and introduced throughconventional hypodermic needles or using infusion pumps.

Both local and systemic administration is contemplated by the invention.Desirable features of local administration include achieving effectivelocal concentrations of the active compound as well as avoiding adverseside effects from systemic administration of the active compound.Localized delivery techniques are described in, for example, 51 J.Biomed. Mat. Res. 96 (2000); 100 J. Control Release 211 (2004); 103 J.Control Release 541 (2005); 15 Vet. Clin. North Am. Equine Pract. 603(1999); 1 Semin. Interv. Cardiol. 17 (1996).

The amount of agent administered to the individual will depend on thecharacteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs as well as the degree, severity andtype of disease as indicated. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors.

Accordingly, with respect to the therapeutic methods of the invention,it is not intended that the administration of a HB-X conjugate, e.g.,HB-X fusion protein comprising a therapeutic protein or peptide belimited to a particular mode of administration, dosage, or frequency ofdosing; the present invention contemplates all modes of administration,including intramuscular, intravenous, intraperitoneal, intravesicular,intraarticular, intralesional, subcutaneous, or any other routesufficient to provide a dose adequate to treat an disease or disorder asdisclosed herein. After formulation with an appropriate pharmaceuticallyacceptable carrier in a desired dosage, a pharmaceutical compositioncomprising HB-X as disclosed herein can be administered to a subject. Apharmaceutical a composition comprising HB-X can be administered to asubject using any suitable means. In general, suitable means ofadministration include, but are not limited to, topical, oral,parenteral (e.g., intravenous, subcutaneous or intramuscular), rectal,intracisternal, intravaginal, intraperitoneal, ocular, or nasal routes.

In a specific embodiment, it may be desirable to administer thepharmaceutical composition comprising HB-X locally to the area in needof treatment; this may be achieved, for example, and not by way oflimitation, by local infusion during surgery, topical application, e.g.,by injection, by means of a catheter, or by means of an implant, theimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, fibers, or commercial skinsubstitutes. In some embodiments, HB-X as disclosed herein are appliedto the muscle using topical creams, patches, intramuscular injectionsand the like.

In some embodiments, HB-X can be administered to a subject orally (e.g.,in capsules, suspensions or tablets) or by parenteral administration.Conventional methods for oral administration include administering HB-Xas tablets, suspensions, solutions, emulsions, capsules, powders, syrupsand the like are usable. Known techniques that deliver HB-X orally orintravenously and retain the biological activity are preferred.Parenteral administration can include, for example, intramuscular,intravenous, intraarticular, intraarterial, intrathecal, subcutaneous,or intraperitoneal administration. HB-X can also be administered orally,transdermally, topically, by inhalation (e.g., intrabronchial,intranasal, oral inhalation or intranasal drops) or rectally.Administration can be local or systemic as indicated. Agents, e.g.,nucleic acid agents which encode HB-X can also be delivered using avector, e.g., a viral vector by methods which are well known to thoseskilled in the art.

When administering a composition comprising HB-X as disclosed hereinparenterally, it will generally be formulated in a unit dosageinjectable form (e.g., solution, suspension, emulsion). Thepharmaceutical formulations suitable for injection include sterileaqueous solutions or dispersions and sterile powders for reconstitutioninto sterile injectable solutions or dispersions. The carrier can be asolvent or dispersing medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol),suitable mixtures thereof, and vegetable oils.

The term “Dosage unit” form as used herein refers to physically discreteunits suited as unitary dosages for the mammalian subjects to betreated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the HB-X as disclosedherein and the particular therapeutic or prophylactic effect to beachieved, and (b) the limitations inherent in the art of compounding aHB-X as an active for the treatment of sensitivity in individuals. Thepharmaceutically acceptable compositions comprising HB-X as disclosedherein can be suspended in aqueous vehicles and introduced throughconventional hypodermic needles or using infusion pumps.

The methods described herein may be used to deliver HB-X to cells, e.g.,human cells, in vitro or ex vivo. Alternatively, the method ofadministering HB-X can be performed on cells present in a subject aspart of an in vivo (e.g., therapeutic or prophylactic) protocol. Forexample, the method can be used to treat or prevent a IGF-1-mediatedindication in a subject, such as therapy for cartilage regenerationfollowing injury. Accordingly, the invention provides a method oftreating (e.g., curing, suppressing, ameliorating, delaying orpreventing the onset of, or preventing recurrence or relapse of) orpreventing permanent cartilage loss. The method includes administeringto a subject a HB-X composition in an amount sufficient to inhibit orreduce cartilage loss or increase cartilage regeneration, therebytreating or preventing joint degeneration in a subject.

Pharmaceutical Compositions

The compositions of the present invention can be contained inpharmaceutically acceptable formulations. Such a pharmaceuticallyacceptable formulation may include a pharmaceutically acceptablecarrier(s) or excipient(s), solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, that are physiologically compatible. For example,the carrier can be suitable for intra-articular injection. Excipientsinclude pharmaceutically acceptable stabilizers. The present inventionpertains to any pharmaceutically acceptable formulations, includingsynthetic or natural polymers in the form of macromolecular complexes,nanocapsules, microspheres, or beads, and lipid-based formulationsincluding oil-in-water emulsions, micelles, mixed micelles, syntheticmembrane vesicles, and gels such as hyaluronic gels.

In some embodiments, a composition comprising HB-X as disclosed hereincan be formulated in any suitable means, e.g., as a sterile injectablesolution, e.g., which can be prepared by incorporating the HB-X in therequired amount of the appropriate solvent with various of the otheringredients, as desired. In some embodiments, a composition comprisingHB-X as disclosed herein can be formulated in a hydrogel, for example,but not limited to a hydrogel comprising self-assembling peptides isRADA-16 (also known as PURAMATRIX®) and KLD-12.

A pharmacological formulation of a composition comprising HB-X asdisclosed herein can be administered to the patient in an injectableformulation containing any compatible carrier, such as various vehicles,adjuvants, additives, and diluents; or the compounds utilized in thepresent invention can be administered parenterally to the patient in theform of slow-release subcutaneous implants or targeted delivery systemssuch as monoclonal antibodies, vectored delivery, iontophoretic, polymermatrices, liposomes, and microspheres. Examples of delivery systemsuseful in the present invention include those presented in U.S. Pat.Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603;4,486,194; 4,447,233; 4,447, 224; 4,439,196 and 4,475,196. Other suchimplants, delivery systems, and modules are well known to those skilledin the art.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, e.g., parabens,chlorobutanol, phenol and sorbic acid. In many cases, it will bedesirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the compounds.

In another embodiment, a composition comprising HB-X as disclosed hereincan comprise lipid-based formulations. Any of the known lipid-based drugdelivery systems can be used in the practice of the invention. Forinstance, multivesicular liposomes, multilamellar liposomes andunilamellar liposomes can all be used so long as a sustained releaserate of the encapsulated active compound can be established. Suchformulations may be used to further modify the release profile of theHB-X composition. Methods of making controlled release multivesicularliposome drug delivery systems are described in PCT ApplicationPublication Nos: WO 9703652, WO 9513796, and WO 9423697, the contents ofwhich are incorporated herein by reference.

The composition of the synthetic membrane vesicle is usually acombination of phospholipids, usually in combination with steroids,especially cholesterol. Other phospholipids or other lipids may also beused. Examples of lipids useful in synthetic membrane vesicle productioninclude phosphatidylglycerols, phosphatidylcholines,phosphatidylserines, phosphatidylethanolamines, sphingolipids,cerebrosides, and gangliosides, with preferable embodiments includingegg phosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidyleholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.

In preparing lipid-based vesicles containing HB-X, such variables as theefficiency of active compound encapsulation, labiality of the activecompound, homogeneity and size of the resulting population of vesicles,active compound-to-lipid ratio, permeability, instability of thepreparation, and pharmaceutical acceptability of the formulation shouldbe considered.

In another embodiment, the HB-X can be delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533). In yetanother embodiment, HB-X can be delivered in a controlled releasesystem. In one embodiment, a pump may be used (see Langer (1990) supra).In another embodiment, polymeric materials can be used (see Howard etal. (1989) J. Neurosurg. 71: 105). In another embodiment where theactive agent of the invention is a nucleic acid encoding HB-X, thenucleic acid can be administered in vivo to promote expression of itsencoded protein, by constructing it as part of an appropriate nucleicacid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see, for example,U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88: 1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

Prior to introduction, a composition comprising HB-X as disclosed hereincan be sterilized, by any of the numerous available techniques of theart, such as with gamma radiation or electron beam sterilization.

In another embodiment of the invention, a composition comprising HB-X orvariant thereof as disclosed herein, can be administered and/orformulated in conjunction (e.g., in combination) with any othertherapeutic agent. For purpose of administration, HB-X as disclosedherein is preferably formulated as a pharmaceutical composition.Pharmaceutical compositions of the present invention comprise a compoundof this invention and a pharmaceutically acceptable carrier, wherein thecompound is present in the composition in an amount which is effectiveto treat the condition of interest. Appropriate concentrations anddosages can be readily determined by one skilled in the art.

Pharmaceutically acceptable carriers are familiar to those skilled inthe art. For compositions formulated as liquid solutions, acceptablecarriers include saline and sterile water, and may optionally includeantioxidants, buffers, bacteriostats and other common additives. Thecompositions can also be formulated as pills, capsules, granules, ortablets which contain, in addition to a compound of this invention,diluents, dispersing and surface active agents, binders, and lubricants.One skilled in this art may further formulate the compounds of thisinvention in an appropriate manner, and in accordance with acceptedpractices, such as those disclosed in Remington's PharmaceuticalSciences, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1990.

The compositions of the present invention can be in any form. Theseforms include, but are not limited to, solutions, suspensions,dispersions, ointments (including oral ointments), creams, pastes, gels,powders (including tooth powders), toothpastes, lozenges, salve, chewinggum, mouth sprays, pastilles, sachets, mouthwashes, aerosols, tablets,capsules, transdermal patches, that comprise one or more resolvinsand/or protectins or their analogues of the invention.

Formulations of a composition comprising HB-X as disclosed herein can beprepared by a number or means known to persons skilled in the art. Insome embodiments the formulations can be prepared for administration asan aerosol formulation, e.g., by combining (i) HB-X as disclosed hereinin an amount sufficient to provide a plurality of therapeuticallyeffective doses; (ii) the water addition in an amount effective tostabilize each of the formulations; (iii) the propellant in an amountsufficient to propel a plurality of doses from an aerosol canister; and(iv) any further optional components e.g. ethanol as a cosolvent; anddispersing the components. The components can be dispersed using aconventional mixer or homogenizer, by shaking, or by ultrasonic energy.Bulk formulation can be transferred to smaller individual aerosol vialsby using valve to valve transfer methods, pressure filling or by usingconventional cold-fill methods. It is not required that a stabilizerused in a suspension aerosol formulation be soluble in the propellant.Those that are not sufficiently soluble can be coated onto the drugparticles in an appropriate amount and the coated particles can then beincorporated in a formulation as described above.

In certain embodiments, a composition comprising HB-X, which is anucleic acid agent or polypeptide agent can be administered to a subjectas a pharmaceutical composition with a pharmaceutically acceptablecarrier. In certain embodiments, these pharmaceutical compositionsoptionally further comprise one or more additional therapeutic agents.In certain embodiments, the additional therapeutic agent or agents areautoimmune disease or drugs, such as immune suppressants and the like.Of course, such therapeutic agents are which are known to those ofordinary skill in the art can readily be substituted as this list shouldnot be considered exhaustive or limiting.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred percent,this amount will range from about 1 percent to about ninety-nine percentof active ingredient, preferably from about 5 percent to about 70percent, most preferably from about 10 percent to about 30 percent.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste. Insolid dosage forms of the invention for oral administration (capsules,tablets, pills, dragees, powders, granules and the like), the activeingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof. Besides inert diluents, theoral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, coloring,perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

In some instances, a composition comprising HB-X as disclosed herein canbe in a formulation suitable for rectal or vaginal administration, forexample as a suppository, which may be prepared by mixing one or morecompounds of the invention with one or more suitable nonirritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax or a salicylate, and which issolid at room temperature, but liquid at body temperature and, thereforerelease the active compound. Suitable carriers and formulations for suchadministration are known in the art.

Dosage forms for the topical or transdermal administration of HB-X,e.g., for muscular administration include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. HB-X asdisclosed herein may be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof. Powders and sprays cancontain, in addition to a compound of this invention, excipients such aslactose, talc, silicic acid, aluminum hydroxide, calcium silicates andpolyamide powder, or mixtures of these substances. Sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of HB-X to the body. Such dosage forms can be made bydissolving or dispersing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate of such flux can be controlled by either providing arate controlling membrane or dispersing the active compound in a polymermatrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto further modify the absorption of the drug from subcutaneous orintramuscular injection beyond the modification accomplished through HBdomain fusion. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

In certain embodiments, HB-X can be isolated and/or purified orsubstantially purified by one or more purification methods describedherein or known by those skilled in the art. Generally, the purities areat least 90%, in particular 95% and often greater than 99%. In certainembodiments, the naturally occurring compound is excluded from thegeneral description of the broader genus.

In some embodiments, the composition comprises at least one HB-X incombination with a pharmaceutically acceptable carrier. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, without limitation: sugars, such as lactose, glucose andsucrose; starches, such as corn starch and potato starch; cellulose, andits derivatives, such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients, such as cocoa butter and suppository waxes; oils, such aspeanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, cornoil and soybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations.

In certain embodiments, a composition comprising HB-X as disclosedherein can contain one or more acidic functional groups and, thus, arecapable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts, esters, amides, and prodrugs” as used herein refers to thosecarboxylate salts, amino acid addition salts, esters, amides, andprodrugs of the compounds of the present invention which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of patients without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit/riskratio, and effective for their intended use of the compounds of theinvention. The term “salts” refers to the relatively non-toxic,inorganic and organic acid addition salts of compounds of the presentinvention.

These salts can be prepared in situ during the final isolation andpurification of the compounds or by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. These may include cations based onthe alkali and alkaline earth metals, such as sodium, lithium,potassium, calcium, magnesium and the like, as well as non-toxicammonium, quaternary ammonium, and amine cations including, but notlimited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. (See, for example, Berge S. M., et al., “PharmaceuticalSalts,” J. Pharm. Sci., 1977; 66: 1-19 which is incorporated herein byreference).

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. The term isfurther intended to include lower hydrocarbon groups capable of beingsolvated under physiological conditions, e.g., alkyl esters, methyl,ethyl and propyl esters.

As used herein, “pharmaceutically acceptable salts or prodrugs” aresalts or prodrugs that are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use.These compounds include the zwitterionic forms, where possible, of rcompounds of the invention.

The term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the present invention. Thesesalts can be prepared in situ during the final isolation andpurification of the compounds or by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. These may include cations based onthe alkali and alkaline earth metals, such as sodium, lithium,potassium, calcium, magnesium and the like, as well as non-toxicammonium, quaternary ammonium, and amine cations including, but notlimited to ammonium, tetramethylammonium, tetraethyl ammonium, methylamine, dimethyl amine, trimethylamine, triethylamine, ethylamine, andthe like (see, e.g., Berge S. M., et al. (1977) J. Pharm. Sci. 66, 1,which is incorporated herein by reference).

The term “prodrug” refers to compounds or agents that are rapidlytransformed in vivo to yield the active HB-X, e.g., a biologicallyactive or functional active HB-X which encodes a functionally activetherapeutic peptide or protein. In some embodiments, HB-X prodrugs canbe activated by hydrolysis in blood, e.g., via cleavage of a precursortherapeutic protein into an active therapeutic protein, similar to howinsulin is activated from its proprotein into an active insulin protein.A thorough discussion is provided in T. Higachi and V. Stella,“Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. SymposiumSeries, and in Bioreversible Carriers in: Drug Design, ed. Edward B.Roche, American Pharmaceutical Association and Pergamon Press, 1987,both of which are hereby incorporated by reference. As used herein, aprodrug is a compound that, upon in vivo administration, is metabolizedor otherwise converted to the biologically, pharmaceutically ortherapeutically active form of the compound. The prodrug may be designedto alter the metabolic stability or the transport characteristics ofHB-X, to mask side effects or toxicity, or to alter othercharacteristics or properties of HB-X. By virtue of knowledge ofpharmacodynamic processes and drug metabolism or post-translationalprotein processing of HB-X in vivo, once a pharmaceutically activecompound is identified, those of skill in the pharmaceutical artgenerally can design HB-X prodrugs which can be activated in vivo toincrease levels of the therapeutic protein present in HB-X in thesubject (see, e.g., Nogrady (1985) Medicinal Chemistry A BiochemicalApproach, Oxford University Press, N.Y., pages 388-392). Conventionalprocedures for the selection and preparation of suitable prodrugs aredescribed, for example, in “Design of Prodrugs,” ed. H. Bundgaard,Elsevier, 1985. Suitable examples of prodrugs include methyl, ethyl andglycerol esters of the corresponding acid.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of ordinary skill in the art.

Vectors

In another embodiment, this invention provides vector encoding HB-Xfusion proteins for use in the methods, compositions and kits asdisclosed herein. In some embodiments, the vector is an expressionvector and enables the insertion of the nucleic acid sequence encodingan active agent of an investigators choice. In some embodiments, thepresent invention relates to a vector comprising a nucleic acid encodingat least one heparin binding peptide (HB) selected fromKRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1), KRKKKGKGLGKKRDPRLRKYK (SEQ ID NO:2), or KRKKKGKGLGKKRDPKLRKYK (SEQ ID NO: 3). In some embodiments, thevector comprises a multiple cloning site for insertion a nucleic acidsequence encoding an active agent of an investigators choice at the 3′or 5′ or both of the HB sequence. In some embodiments, the vectorcomprises multiple HB peptides. In some embodiments, the vector alsocomprises at least one nucleic acid encoding a linker peptide (e.g.,comprising at least GGG) at the 3′ or 5′ of the HB peptide sequence,before the multiple cloning site, depending on whether the nucleic acidsequence encoding X is inserted at the 3′ or 5′ or both of the HBpeptide nucleic acid sequence. In some embodiments, the vector alsocomprises at least one nucleic acid sequence encoding at least oneactive agent (X).

In some embodiments, the vector comprises a nucleic acid sequence whichencodes at least one HB-Xn or Xn-HBn fusion protein, wherein HB is aheparin binding peptide selected from KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO:1), KRKKKGKGLGKKRDPRLRKYK (SEQ ID NO: 2), or KRKKKGKGLGKKRDPKLRKYK (SEQID NO: 3); X is an active agent, and n is an integer of at least 1.

In some embodiments, the vector comprises a nucleic acid sequence whichencodes at least one (HB-linker)n-Xn or at least one Xn.(HB-linker)nfusion protein, or at least one (HB-linker)n-Xm-(HB-linker)o fusionprotein, wherein HB is a heparin binding peptide selected fromKRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1), KRKKKGKGLGKKRDPRLRKYK (SEQ ID NO:2), or KRKKKGKGLGKKRDPKLRKYK (SEQ ID NO: 3); X is an active agent,wherein m is an integer of at least 1, and n+o is an integer of at least1.

Kits

In another embodiment, this invention provides kits for the practice ofthe methods of this invention. The kits preferably include one or morecontainers containing a HB peptide and means to attach the HB peptide toan active agent (X). In some embodiments, a kit comprises (i) at leastone HB peptide selected from the group consisting of:KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1), KRKKKGKGLGKKRDPRLRKYK (SEQ ID NO:2), or KRKKKGKGLGKKRDPKLRKYK (SEQ ID NO: 3); and a chemical linker toconjugate the HB peptide to a small molecule.

In some embodiments, a kit can comprise a vector comprising a nucleicacid encoding at least one heparin binding peptide (HB) selected fromKRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1), KRKKKGKGLGKKRDPRLRKYK (SEQ ID NO:2), or KRKKKGKGLGKKRDPKLRKYK (SEQ ID NO: 3) as disclosed herein, andsuitable reagents (e.g., restriction enzymes and ligation enzymes etc.)for subcloning a nucleic acid sequence encoding at least one an activeagent into the vector. In some embodiments, the vector in the kit alsocomprises nucleic acid sequences encoding linker peptides, which may be3′ or 5′ (or both) of the nucleic acid sequence encoding the HBpeptides, depending on where the active agent is to be cloned. Inanother embodiment, a kit may comprise a HB-X conjugate, where X is atherapeutic protein or peptide for treatment of a disease or condition,e.g., a cartilage-related disease or disorder, a neurological disorder,an eye disorder or inflammation.

A kit may optionally contain additional therapeutics to beco-administered with the HB-X conjugate. The kit may compriseinstructions for administration of a HB-X conjugate to a subject with anallergy, cartilage-related disease or disorder, a neurological disorder,an eye disorder, inflammation, or to establish immunity throughvaccination.

The kits may also optionally include appropriate systems (e.g. opaquecontainers) or stabilizers (e.g. antioxidants) to prevent degradation ofthe HB-X conjugate by light or other adverse conditions.

In another aspect of the invention provides kits including one or morecontainers containing a HB-X conjugate as disclosed herein and apharmaceutically acceptable excipient. The kit may optionally containadditional therapeutics to be co-administered with the HB-X conjugate.The kit may comprise instructions for administration of a subject withan allergy, cartilage-related disease or disorder, a neurologicaldisorder, an eye disorder, inflammation, or to establish immunitythrough vaccination.

The kits may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of HB-X conjugatesfor the treatment of a disease in a mammal, e.g., for the treatment ofan allergy, cartilage-related disease or disorder, a neurologicaldisorder, an eye disorder, inflammation, or to establish immunitythrough vaccination.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

Cartilage Binding Assay

Material and Methods

Cartilage explants were harvested from stifle joints of 1-2-week-oldnewborn bovine calves (Research 87 Inc., Boylston Mass.). Articularcartilage was sliced and punched into 1-mm-thick by 3-mm-diameter disksand incubated at 37 C in low-glucose DMEM.

Cartilage disks were incubated with medium alone, PTH, or eHB-PTH. After24 hours, all cartilage disks were washed×3 in fresh medium with noadded peptide. Cartilage was then returned to incubation and harvestedafter two days or four days in the absence of peptide.

Protein Extraction from Cartilage Discs and Analysis:

Cartilage disks were individually pulverized while cooled with liquidnitrogen. The powder was resuspended in lysis buffer containing 0.1%Triton X-100, 1 mM PMSF and protease inhibitor cocktail (Sigma) androtated overnight at 4° C. The resulting extracts were clarified bycentrifugation and protein concentration assayed with 660 nm ProteinAssay (Pierce).

For Western analysis, portions of extracts containing equal amounts ofprotein (3 ug total protein/lane) were boiled under reducing conditionsand electrophoresed on 4-12% Bis-Tris gels. A rabbit polyclonal anti-PTHantibody was from Abcam (ab40630) for western blotting analysis of thepresence of PTH in the cartilage explants. Membranes were probed for PTHwith a 1:1000 dilution of antibody ab40630, followed by a secondary goatanti-rabbit antibody at a dilution of 1:5000.

For Assessment of HB-IGF1 Retention in the Spinal Cord:

Retired male breeder rats were obtained from Charles River. A rat waseuthanized and tissues were harvested. Spinal cord tissue was takenafter dissection and divided into portions of similar. Each portion wasweighed and then incubated in 1 mL serum-free DMEM at 37 C in a 24-wellplate. Medium was then replaced with serum-free DMEM containing eitherno additions, 1 ug/ml of IGF-1, or 1 ug/ml HB-IGF-1. IGF-1 was humanrecombinant IGF-1 (Increlex, Tercica). IGF-1 was fused to HB(C17R) (SEQID NO: 21). HB-IGF-1 was expressed as human recombinant HB-C17R-IGF-1,and extracted from inclusion bodies after expression in E. coli. Afterincubation of the spinal cord with the HB-IGF1 for one day, medium wasreplaced with serum-free DMEM containing no additions. One set of tissuewas collected as the “Day 0” samples and stored frozen at −20 C. Tissuesamples were then taken after 24 hours of wash-out (“Day 1”).

Protein Extraction from the Spinal Cord and Analysis:

Proteins were extracted from the frozen spinal cord tissues byhomogenizing in 1 ml lysis buffer/100 mg tissue. The lysis buffercontained 0.1% Triton X-100 with 1 mM PMSF and protease inhibitorcocktail (Sigma). Total protein concentrations were measured andportions of extracts containing equivalent amounts of total protein wereboiled in reducing sample buffer and loaded on 4-12%) Bis-Tris gels forWestern analysis.

Determination of Expression of Fusion Proteins Comprising C17K and C17RHB Peptides:

Superior Expression of Soluble HB-IGF-1 was detected with enhanced HBpeptides (e.g., C17R and C17K) sequences as compared to wildtype (SEQ IDNO: 20) and C17S sequences (SEQ ID NO: 41). The protein expression fromfollowing plasmids were assessed: (i) Plasmid 04 HB(C17K)-IGF-1 inpET24a(+), (ii) Plasmid 05 HB(C17R)-IGF-1 in pET24a(+), (iii) plasmid 06HB(C17S)-IGF-1 in pET24a(+), and (iv). Plasmid 07: HB-IGF-1 inpET24a(+). HB-IGF-1 and mutants (listed above) were transformed into T7Express E. coli cells and grown in Luria-Bertani (LB) medium in 1 Lbatches. Protein expression was induced with 1 mM isopropylβ-D-thiogalactoside for 4 h, and cells were then harvested bycentrifugation. Proteins were extracted with 10 ml BugBuster Master Mixnative extraction reagent (Novagen). 10 ul of each sample was run on a4-12% Bis-Tris gel in reducing conditions. Expression levels werecompared by western blot using IGF-1 antibody ab9572 (AbCam).

Determination of Yield of Production of Fusion Proteins Comprising C17Kand C17R HB Peptides:

A superior yield on purification of soluble HB-IGF-1 fusion proteinscomprising enhanced HB (C17R) peptide was detected. The following yieldof fusion protein from the following plasmids were assessed: (i) Plasmid05 HB(C17R)-IGF-1 in pET24a(+) or (ii) Plasmid 07: HB-IGF-1 inpET24a(+).

HB-IGF-1 and HB(C17R)-IGF-1 (listed above) were transformed into T7Express E. coli cells and grown in Luria-Bertani (LB) medium in 1 Lbatches. Protein expression was induced with 1 mM isopropyl13-D-thiogalactoside for 4 h, and cells were then harvested bycentrifugation. Proteins were extracted with BugBuster Master Mix nativeextraction reagent (Novagen). Purification was performed by loadingsamples onto a HiLoad 16/60 Superdex 200 size exclusion chromatographycolumn. 2 ml was eluted in each fraction. 10 ul of each fraction was runon a 4-12% Bis-Tris gel in reducing conditions. IGF-1 in each fractionwas assayed for by western blot using IGF-1 antibody ab9572 (AbCam).Protein was detected in fractions 3-12 (fractions shown) for the C17Rvariant (eHB) and in fractions 4-10 for the wild type variant.

To demonstrate that enhanced HB (C17R) allows superior yield of HB-IGF-1fusion protein from inclusion bodies, plasmids encoding HB-IGF-1 andHB(C17R)-IGF-1 (e.g., Plasmid 05 HB(C17R)-IGF-1 in pET24a(+) or (ii)Plasmid 07: HB-IGF-1 in pET24a(+) as listed above) were transformed intoT7 Express E. coli cells and grown in Luria-Bertani (LB) medium in 1 Lbatches. For induced samples, protein expression was induced with 1 mMisopropyl β-D-thiogalactoside for 4 h. Non-induced samples were allowedto grow for 4 h without 1 mM isopropyl β-D-thiogalactoside. Cells werethen harvested by centrifugation. Cells were lysed in 8 ml lysis buffercontaining 6 M guanidine hydrochloride, 20 mM sodium phosphate, 500 mMNaCl, pH 7.8. Cell lysates were dialyzed into a buffer containing 50 mMTris, 100 mM NaCl. 5 ul of each sample was run on a 4-12% Bis-Tris gelin reducing conditions. Samples were analyzed for IGF-1 by western blotusing IGF-1 antibody ab9572 (AbCam) and by Coomassie stain.

Example 1 HB-IGF-1 Fusion Protein

A HB-IGF-1 construct was made to express a heparin-binding domain fusedto the amino-terminus of a mature IGF-1 protein. The HB-IGF-1 fusionprotein was produced by recombinant expression in E. coli, refolded, andpurified by reverse-phase chromatography. Human recombinant IGF-1 isavailable commercially, for example INCRELEX® (mecasermin [rDNA origin],Ipsen Biopharmaceuticals, Inc., Basking Ridge, N.J.).

Example 2 In Vivo Binding and Pharmacokinetics

Experiments were performed with male Lewis rats (251-275 g, CharlesRiver, Wilmington, Mass.). All animal procedures were approved by theHarvard Medical Area Standing Committee on Animals.

Rats received a single intraarticular injection containing 100 μg ofHB-IGF-1, 100 μg IGF-1, or phosphate buffered saline (PBS) in the rightpatellofemoral joint. Articular cartilage, medial meniscus and patellartendon samples were harvested at 2, 4, 6, and 8 days after injection.Samples were weighed, pulverized while in liquid nitrogen and extractedwith 10 μl of lysis buffer (100 mM NaCl, 50 mM Tris, 0.5% Triton X-100,5 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail [Roche]) permilligram of tissue. Portions of extracts with equal protein mass wereanalyzed by Western blotting. Serum IGF-1 levels were measured by ELISA(R&D Systems #DY291) reactive with human but not rodent IGF-1.

Example 3 Cartilage Biosynthesis Assay

Rats were randomly assigned to receive a single intraarticular injectioncontaining 100 μg of HB-IGF-1, 100 μg IGF-1, or PBS in the rightpatellofemoral joint. Animals were sacrificed 2 or 4 days afterinjection. Following sacrifice, the meniscus from the right knee jointwas harvested and incubated at 37° C. in Dulbecco's Modified EagleMedium (DMEM) containing 5 μCi/ml 35S-sulfate for 18 hr.

Following incubation, samples were washed four times for 15 min in PBSwith sulfate to remove unincorporated radiolabel. Samples were digestedovernight with 1 mg/ml Proteinase K at 60° C. and radiolabelincorporation was measured in a liquid scintillation counter.

Example 4 Rat Model of Joint Damage

For surgical procedures, rats were randomly assigned to one of threegroups: 50 μA intraarticular injections containing 100 μg of HB-IGF-1,100 μg IGF-1, or PBS in the right knee joint. Initial injections wereadministered 1 day prior to medial meniscal tear (MMT). The MMT modelwas used as previously described. Gerwin et al., 18 Osteoarthr. Cartil.S24 (2010). Briefly, a skin incision was made across the medial aspectof the knee. The medial collateral ligament was exposed by bluntdissection and transected. The medial meniscus was reflected mediallyand cut to simulate a full tear. Subsequent intraarticular injectionswere administered 7 and 14 days post MMT. Animals were sacrificed 21days after surgery.

Histological staging and sectioning was performed. Knee joints wereharvested and fixed in 4% paraformaldehyde. Joints were then transferredto 5% formic acid decalcifying solution (ImmunoCal, Decal Chemical Corp,Tallman, N.Y.). Joints were cut in half to form anterior and posteriorsections, and embedded in paraffin. 8 μm sections taken approximately200 μm apart were stained with Toluidine Blue. The medial tibial plateauwas analyzed and imaged microscopically. The central most sectionsexhibiting the maximum injury extent were selected for blinded scoring.Injuries were scored using a modified Mankin scoring system. Injurieswere measured using three different metrics: cartilage matrix losswidth, total cartilage degeneration width, and significant degenerationwidth.

Cartilage matrix loss width measured only the extent of 100% matrix losswhile areas of PG or chondrocyte degeneration are ignored. Measurementswere taken at the surface (0% depth) and at the tidemark (100%) depth.Total cartilage degeneration width measured the total width of the areaof articular cartilage affected by any type of degenerative change.Significant cartilage degeneration width measured the extent of injurythat affects more than 50% of the thickness of cartilage. Significantcartilage degeneration width included any form of collagen matrix, PG,or chondrocyte degeneration. All results are expressed as mean±SEM.

Example 5 Increased Expression and Yield of HB-IGF-1 in E. coli

The inventors demonstrated that modification of amino acid residue 17 inSEQ ID NO: (corresponding to amino acid residue 16 in SEQ ID NO: 1)could increase the expression of the HB-fusion protein in E. coli. Theinventors demonstrate that there is unexpected superior expression ofsoluble HB-IGF-1 from E. coli expressing fusion proteins comprisingenhanced HB (eHB) peptides: C17K (SEQ ID NO: 22) and C17R (SEQ ID NO:21), as compared to HB-IGF1 fusion proteins comprising C17S(MKRKKKGKGLGKKRDPSLRKYK; SEQ ID NO: 41) or wild-type HB (SEQ ID NO: 20)(FIG. 5).

Furthermore, surprisingly, the inventors detected a significantly higheryield upon purification of soluble HB-IGF-1 from E. coli expressingHB-IGF-1 fusion proteins comprising C17R (SEQ ID NO: 21) as compared toE. coli expressing an HB-IGF-1 fusion protein comprising wild-type HB(SEQ ID NO: 20) (FIG. 6).

Next, the inventors also demonstrate that a higher yield of HB-IGF-1 canbe obtained from the inclusion bodies produced by E. coli expressing anHB-IGF-1 fusion protein comprising C17R (SEQ ID NO: 21) as compared toE. coli expressing an HB-IGF-1 comprising wild-type HB (SEQ ID NO: 20)(FIG. 7).

Accordingly, the inventors demonstrate that modification of an unchargedresidue in SEQ ID NO: 1 or SEQ ID NO: 20, e.g., a change of the cysteineresidue at amino acid 17 in SEQ ID NO: 20, (or a change of the cysteineamino acid residue 16 in SEQ ID NO: 1) to a positively charged reside(e.g., arginine or lysine) can surprisingly result in a significantincrease in the amount of both the soluble and insoluble HB-fusionprotein expressed in E. coli, resulting in increased yield of theHB-fusion protein both from purified soluble fractions (FIG. 6) and fromextracts of the E. coli inclusion bodies (FIG. 7).

Example 6 Spinal Cord

In Examples 1-4, the inventors demonstrate that that heparin-binding(HB) fusions with IGF-1 allow for extended retention of the fusionprotein within cartilage tissue through interaction with highly abundantchondroitin sulfated proteoglycans. However, other tissues also haveabundant negatively charged proteoglycans in their extracellular matrix.Accordingly, the inventors assessed whether a HB-IGF-1 fusion proteinwould extend retention of the protein in neural tissue harvested fromthe spinal cord.

After incubation of the spinal cord explants in the presence of IGF-1 orHB-IGF-1 for one day, both proteins were detected in the tissue extractsby Western analysis for IGF-1 (FIG. 8). After one day of washout, nodetectable non-fused IGF-1 was detected to be remaining in the tissue.In contrast, the HB-IGF-1 protein remains detectable in the spinal cordtissue extracts (FIG. 8).

Accordingly, the inventors have demonstrated herein that that HB-IGF-1is retained in spinal cord tissue ex vivo for at least 24 hours, whereasunmodified (non-fused) IGF-1 is not. Accordingly, HB fused to a proteinallows extended retention of the proteins in tissues other thancartilage, including neural tissue and other tissues with abundantchondroitin sulfated proteoglycans on cell surfaces of the tissues.

Example 7 PTH

In Examples 1-4, the inventors demonstrate that fusion of the HB domainwith IGF-1 allows for targeted and sustained retention of the IGF-1fusion protein in cartilage. However, as discussed herein, fusion of HBdomain are not limited to the IGF-1 protein. Accordingly, HB can befused to any active agent as a strategy for targeted delivery ofmultiple therapeutic proteins.

A peptide of the parathyroid hormone (PTH), e.g., amino acids 1-34(PTH(1-34) is a peptide approved for clinical use in osteoporosis thathas potential benefits in cartilage repair. In a rat osteoarthritismodel, it has been shown to reduce the extent of osteoarthritisdevelopment (Chang et al., 2009).

As demonstrated herein, the inventors assessed the retention of PTH inthe cartilage using bovine tissue culture explant using a PTH-HB fusionprotein.

The following PTH peptides were generated by synthesis (Peptide 2.0,Chantilly Va.):

1. PTH(1-34)-biotin: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFK(-biotinyl)-NH2(SEQ ID NO: 80)

2. PTH(1-34)-linker-HB-biotin:SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFGGGKRKKKGKGLGKKRDPRLRKYKK(biotinyl)-NH2 (SEQ ID NO: 81).

Biotin was added to the PTH(1-34) and PTH(1-34)-linker-HB peptide foridentification and tracking purposes. Similarly, a PTH(1-34)-linker-HBconstruct without the biotin can be used for therapeutic purposes.Additionally, the absence of a linker can also be used, as disclosedherein. A rabbit polyclonal anti-PTH antibody was from Abcam (ab40630)for western blotting analysis of the presence of PTH in the cartilageexplants.

Western analysis demonstrated detection of both PTH and PTH-HB peptide(FIG. 9A). After two days of incubation in the absence of PTH peptides,there was only faint detection of PTH remaining in the tissue (FIG. 9B).In contrast, there was strong detection of PTH-eHB peptide remaining inthe tissue (FIG. 9B). Furthermore, the PTH-HB peptide remained highlyabundant in the tissue after four days of incubation in the absence ofPTH peptides (data not shown). Again, there was only faint detection ofthe PTH peptide (data not shown).

Accordingly, the inventors have demonstrated herein that modification ofPTH (1-34) peptide by fusion to the heparin binding sequence“KRKKKGKGLGKKRDPRLRKYKK” (SEQ ID NO: 2) allows for extended retention ofthe PTH peptide in cartilage tissue. Accordingly, a PTH-HB peptide canbe retained in the cartilage tissue for preventing cartridge loss afterinjury or during OA.

Example 8 Retention of HB Fusion Proteins in Skin after IntradermalInjection

Rats received an intradermal injection containing 50 μg of HB-IGF-1 or50 μg of HB-IGF-1 (C17S) Skin from the injection site was then harvested6 hours, 12 hours, and 24 hours after injection. Skin was harvested with10 mm biopsy punch and tissue was flash frozen. For protein extraction,skin biopsy samples were pulverized while cooled with liquid nitrogen.The powder was suspended in lysis buffer containing 0.1% Triton X-100, 1mM PMSF and protease inhibitor cocktail (Sigma) and rotated overnight at4° C. The resulting extracts were clarified by centrifugation andprotein concentration assayed with 660 nm Protein Assay (Pierce).

Western blots containing equal amounts of extracted protein (10 m/lanetotal protein) were probed for IGF-1 with a 1:1000 dilution of a rabbitpolyclonal anti-IGF-1 antibody (Abcam ab9572), followed by a secondarygoat anti-rabbit antibody at a dilution of 1:5000 HB-IGF-1 wasdetectable in the tissue after 6 hours, whereas IGF-1 was undetectable(FIG. 10). Additionally, HB-IGF-1 remained detectable in the harvestedtissue for at least 24 hours after injection.

Example 9 Retention of HB Fusion Proteins in Skin after SubcutaneousInjection

Radiolabeled proteins were prepared by mixing [1-14C]-acetic anhydride(ARC0102) with anhydrous dioxane and unlabeled HB-IGF-1 (C17S) or IGF-1(Increlex). Rats were injected with either 100 μg 14C-HB-IGF-1 or 100 μg14C-IGF-1. Following 30 minutes incubation, proteins were dialyzedovernight into a sodium phosphate buffer solution. Tissue was harvested24 hours later with a 6 mm biopsy punch. Tissue samples were digested in100 ng/mL Proteinase K for at least 24 hours at 65° C. Radioactivity ofthe tissue digests was measured by a liquid scintillation counter.

Recovery of radiolabeled protein was more than three-fold higher intissue at the site of injection with HB-IGF-1 compared to IGF-1 (FIG.11).

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

What is claimed is:
 1. A composition for controlled systemic release ofa therapeutic, the composition comprising: a therapeutic moiety; and atleast one heparin binding peptide linked to the therapeutic moiety,wherein the heparin binding peptide includes a portion with a sequenceselected from the group consisting of KRKKKGKGLGKKRDPRLRKYK (SEQ ID NO:2); and KRKKKGKGLGKKRDPKLRKYK (SEQ ID NO: 3).
 2. The composition ofclaim 1 wherein the therapeutic moiety comprises insulin-like growthfactor 1 (IGF-1) or Interleukin 1 receptor antagonist (IL-IRA).
 3. Thecomposition of claim 1, further comprising one or more of apolyhistidine tag, a FLAG-tag, and a hydrogel.
 4. The composition ofclaim 1, wherein the composition is produced through E. coli expression.5. The composition of claim 1, wherein the therapeutic moiety comprisesa Neurotrophic factor.
 6. The composition of claim 5, wherein theNeurotrophic factor is selected from the group consisting ofneurotrophins, glial cell-line derived neurotrophic factor familyligands, and neuropoietic cytokines.
 7. The composition of claim 1,wherein the therapeutic moiety comprises a therapeutic antibody.
 8. Acomposition for increasing retention of a therapeutic in tissue, thecomposition comprising: a therapeutic protein; and at least one heparinbinding peptide linked to the therapeutic protein, wherein the heparinbinding peptide includes a portion with a sequence selected from thegroup consisting of KRKKKGKGLGKKRDPRLRKYK (SEQ ID NO: 2); andKRKKKGKGLGKKRDPKLRKYK (SEQ ID NO: 3).
 9. The composition of claim 8,wherein the therapeutic protein comprises an antigen.
 10. Thecomposition of claim 9, wherein the antigen is an allergen adapted toinduce tolerance in a patient to one-selected from the group consistingof pollen, pet dander, dust mites, airborne molds, tree nuts, peanuts,fruits, milk, eggs, fish, shellfish, honey bee venom, yellow jacketvenom, hornet venom, wasp venom, and fire ant venom.